Controlled Delivery of TLR Agonists in Structural Polymeric Devices

ABSTRACT

The present invention comprises compositions, methods, and devices for creating an stimulating an antigen-specific dendritic cell immune response. Devices and methods provide prophylactic and therapeutic immunity to subjects against cancer and infectious agents.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/741,271, filed Jan.14, 2013, which (i) claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 61/586,624, filed Jan. 13,2012; (ii) is a continuation-in-part of U.S. Ser. No. 12/867,426, filedJan. 13, 2012, which is a national stage application, filed under 35U.S.C. §371, of International Application No. PCT/US2009/000914, filedFeb. 13, 2009, which claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 61/143,630, filed Jan. 9,2009 and U.S. Provisional Application No. 61/065,672, filed Feb. 13,2008; and (iii) is a continuation-in-part of U.S. Ser. No. 13/510,356,filed Nov. 22, 2010, which is a national stage application, filed under35 U.S.C. §371, of International Application No. PCT/US2010/057630,filed Nov. 22, 2010, which claims the benefit of priority under 35U.S.C. §119(e) to U.S. Provisional Application No. 61/281,663, filedNov. 20, 2009. Each of these applications is incorporated herein byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under R37 DE013033awarded by the National Institutes of Health. The Government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web. The contents of the text file named“29297-091D01US_ST25.txt”, which was created on Apr. 21, 2016 and is 35KB in size, are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Dendritic cells (DCs) collect and process antigens for presentation to Tcells. DCs are the most potent activators of the immune system amongantigen presenting cells. Research focused on using dendritic cells fora therapeutic benefit has been slow because dendritic cells are rare anddifficult to isolate.

SUMMARY OF THE INVENTION

The invention features a device and method for stimulating immune cellssuch as dendritic cells, in situ. For example, presentation of Toll-likereceptor (TLR) agonists in the context of the device is used for cancervaccination. Incorporation and presentation of the TLR agonists embeddedin structural polymeric devices specifically stimulates CD8(+) dendriticcells (DCs) (corresponding to CD141+ DCs in humans) and plasmacytoidDCs, which subsets of DCs are critical for cancer vaccination.

Accordingly, the invention provides a device comprising a porouspolymeric structure composition, a tumor antigen, and a toll-likereceptor (TLR) agonist. For example, the device comprises a polymericstructure composition, a tumor antigen, and a combination of toll-likereceptor (TLR) agonists, wherein the TLR agonist is selected from thegroup consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12, and TLR13. For example, the polymericstructure comprises poly (D,L-lactide-co-glycolide) (PLG). Exemplary TLRagonists include pathogen associated molecular patterns (PAMPs), e.g.,an infection-mimicking composition such as a bacterially-derivedimmunomodulator. TLR agonists include nucleic acid or lipid compositions[e.g., monophosphoryl lipid A (MPLA)].

Certain nucleic acids function as TLR agonists, e.g., TLR1 agonists,TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6agonists, TLR7 agonists, TLR8 agonists, TLR9 agonists, TLR10 agonists,TLR11 agonists, TLR12 agonists, or TLR13 agonists. In one example, theTLR agonist comprises a TLR9 agonist such as a cytosine-guanosineoligonucleotide (CpG-ODN), a poly(ethylenimine) (PEI)-condensedoligonucleotide (ODN) such as PEI-CpG-ODN, or double strandeddeoxyribonucleic acid (DNA). TLR9 agonists are useful to stimulateplasmacytoid DCs. For example, the device comprises 5 μg, 10 μg, 25 μg,50 μg, 100 μg, 250 μg, or 500 μg of CpG-ODN.

In another example, the TLR agonist comprises a TLR3 agonist such aspolyinosine-polycytidylic acid (poly I:C), PEI-poly (I:C),polyadenylic-polyuridylic acid (poly (A:U)), PEI-poly (A:U), or doublestranded ribonucleic acid (RNA).

TLR3 agonists are useful to stimulate CD8+ DCs in mice and CD141+ DCs inhumans A plurality of TLR agonists, e.g, a TLR3 agonist such as poly I:Cand a TLR9 agonist such as CpG act in synergy to activate an anti-tumorimmune response. For example, the device comprises a TLR3 agonist suchas poly (I:C) and the TLR9 agonist (CpG-ODN) or a PEI-CpG-ODN.Preferably, the TLR agonist comprises the TLR3 agonist, poly (I:C) andthe TLR9 agonist, CpG-ODN. The combination of poly (I:C) and CpG-ODN actsynergistically as compared to the vaccines incorporating CpG-ODN orP(I:C) alone.

In some cases, the TLR agonist comprises a TLR4 agonist selected fromthe group consisting of lipopolysaccharide (LPS), monophosphoryl lipid A(MPLA), a heat shock protein, fibrinogen, heparin sulfate or a fragmentthereof, hyaluronic acid or a fragment thereof, nickel, an opoid,α1-glycoprotein (AGP), RC-529, murine β-defensin 2, and completeFreund's adjuvant (CFA). In other cases, the TLR agonist comprises aTLR5 agonist, wherein the TLR5 agonist is flagellin. Other suitable TLRagonists include TRL7 agonists selected from the group consisting ofsingle-stranded RNA, guanosine anologs, imidazoqinolines, and loxorbine.

Preferably, the TLR agonist is present at a concentration effective toinduce the local production of interleukin-12 (IL-12) by dendriticcells.

The invention also provides a device comprising a porous polymericstructure composition, a disease-associated antigen, and a toll-likereceptor (TLR) agonist, wherein the TLR agonist preferentially binds toTLR3. In some cases, the polymeric structure composition comprisespoly-lactide-co-glycolide (PLG). The TLR3 agonist is present in anamount to preferentially stimulate CD8+ dendritic cells or CD141+dendritic cells.

Preferably, the TLR agonist comprises a TLR3 agonist. In some cases, theTLR3 agonist comprises polyinosine-polycytidylic acid (poly I:C) orPEI-poly (I:C). For example, the TLR agonist comprises a nucleic acid.In other cases, the TLR agonist further comprises a TLR9 agonist. Forexample, the TLR9 agonist comprises a cytosine-guanosine oligonucleotide(CpG-ODN) or a PEI-CpG-ODN. Optionally, the device comprises acombination of TLR agonists, the combination comprising a TLR3 agonistand a TLR9 agonist. For example, the TLR3 agonist comprises poly (I:C)and the TLR9 agonist comprises CpG-ODN.

Alternatively, the device comprises a combination of TLR agonists, thecombination comprising a TLR3 agonist and a TLR4 agonist. For example,the TLR3 agonist comprises poly (I:C) and the TLR4 agonist comprisesMPLA.

Optionally, the device further comprises a recruitment composition.Exemplary recruitment compositions include granulocyte macrophage colonystimulating factor (GM-CSF), Flt3L, and CCL20. For example, therecruitment composition comprises encapsulated GM-CSF.

In some cases, the disease-associated antigen comprises a tumor antigen.For example, the tumor antigen comprises a tumor lysate, purifiedprotein tumor antigen, or synthesized tumor antigen.

Optionally, the TLR agonist further comprises pathogen associatedmolecular patterns (PAMPs). For example, the PAMP comprises amonophosphoryl lipid A (MPLA).

Also provided is a device comprising a polymeric structure composition,a tumor antigen, and a combination of TLR agonists, wherein the TLRagonist is selected from the group consisting of TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13.

A method for eliciting an anti-tumor immune response is carried out bycontacting or implanting into a subject a device comprising a polymericstructure composition, a tumor antigen, and a TLR agonist, wherein theTLR agonist preferentially binds to TLR3. For example, the TLR agonistcomprises a TLR3 agonist. Alternatively, the TLR agonist comprises aTLR3 agonist and a TLR9 agonist.

Preferably, the anti-tumor immune response comprises activation of aCD8+ dendritic cell or a CD141+ dendritic cell. In some cases, theanti-tumor immune response comprises activation of a plasmacytoiddendritic cell or a CD141+ dendritic cell. Alternatively, the anti-tumorimmune response comprises a reduction in tumor burden.

Preferably, the TLR agonist is present at a concentration effective toinduce production of interleukin-12 (IL-12) by dendritic cells.

Optionally, the device further comprises granulocyte macrophage colonystimulating factor (GM-CSF). In some examples, the GM-CSF isencapsulated. Another optional recruitment composition is a cytokine.For example, the device comprises 1 μg, 3 μg, 5 μg, 10 μg, 25 μg, or 50μg of GM-CSF.

The device also contains a tumor antigen, e.g., in the form of a tumorlysate (cultured cells or patient-derived primary cells) or purifiedtumor antigen such as a synthesized/synthetic recombinant protein orbiochemically-purified antigen from a tumor cell.

Also with in the invention is a method for eliciting an anti-tumorimmune response by contacting a subject, e.g., implanting into asubject, a device comprising a porous polymeric structure composition, atumor antigen, and a TLR agonist. For example, the TLR agonist isselected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. The devicedescribed above is associated with advantages over earlier vaccines. Themost significant advantage is its ability to stimulate critical subsetsof DCs that mediate potent anti-tumor activity. The method involvesadministering to a subject a device that contains a TLR3 agonist and/ora TLR9 agonist, which leads to elicitation of an anti-tumor immuneresponse characterized by activation of plasmacytoid DCs and/or CD141+DCs in the subject to which the vaccine was administered. The vaccine isuseful for prophylaxis as well as therapy.

The device is administered, e.g., topically applied or implanted, and ispresent over a period of time, e.g., in-dwelling, while constantlyrecruiting, educating, and dispersing or sending cells forth to lymphnodes or sites of disease or infection in the body Improvements overexisting devices include long term, ongoing activation of cells thatenter the device and concomitant long term, ongoing egress ofimmunologically activated, e.g., antigen primed cells. The deviceincludes a scaffold composition, a recruitment composition, and adeployment composition. The deployment composition that mediatesprolonged and continuous egress of primed cells is aninfection-mimicking composition such as a bacterially-derivedimmunomodulator. In preferred embodiments, the bacterially-derivedimmunomodulator is a nucleic acid such as a cytosine-guanosineoligonucleotide (CpG-ODN).

The methods are used to treat a wide variety of diseases and to developvaccines against a wide variety of antigens. In a preferred embodiment,the present invention is used to develop a cancer vaccine. Anotherpreferred embodiment of the present invention comprises aninfection-mimicking microenvironment with means to activate the hostimmune system and subsequently induce an immune response. The use of asynthetic cytosine-guanosine oligodeoxynucleotide (CpG-ODN) sequencewith exogenous granulocyte macrophage colony stimulating factor (GM-CSF)provides a method for precisely controlling dendritic cell migration andmodulating antigen-specific immune responses. In fact, the approach ofusing of this synthetic cytosine-guanosine oligonucleotide (CpG-ODN)sequence and/or poly (I:C) demonstrates significant improvements overearlier immune therapies.

Various components of the device are tabulated and described below.

TABLE 1 FUNCTION Present an Induce DC EXEMPLARY Attract a DC toImmunogenic Migration from DEVICE Device Factor Device 1 ScaffoldScaffold Scaffold Composition Composition Composition 2 BioactiveBioactive Bioactive Composition Composition Composition 3 ScaffoldBioactive Bioactive Composition Composition Composition 4 ScaffoldScaffold Bioactive Composition Composition Composition 5 BioactiveScaffold Scaffold Composition Composition Composition 6 BioactiveBioactive Scaffold Composition Composition Composition 7 BioactiveScaffold Bioactive Composition Composition Composition 8 ScaffoldBioactive Scaffold Composition Composition Composition

Devices perform three primary functions, e.g. attracting cells to thedevice, presenting an immunogenic factor, and inducing cell migrationaway from the device. Each of these primary functions are performed bythe scaffold (bold font) and/or biological (standard font)composition(s). Table 1 provides exemplary combinations of either thescaffold or biological composition paired with at least one primaryfunction in exemplary devices (1-8). For example, the scaffoldcomposition performs all three primary functions (device 1). In analternative example, the scaffold composition performs one primaryfunction, e.g. attracts cells to the device (preferably, dendriticcells), whereas the biological composition performs two primaryfunctions, e.g. presents an immunogenic factor and induces cells(preferably, dendritic cells) to migrate away from the device (device3). Device 5, for instance, is the inverse combination of device 3.Exemplary secondary functions of the scaffold and/or biologicalcompositions include, but are not limited to, targeting the device to aparticular cell or tissue type, adhering/releasing the device to/fromthe surface of one or more cells or tissues, and modulating thestability/degradation of the device.

The invention comprises a device comprising a scaffold composition andbioactive composition, the bioactive composition being incorporated intoor conjugated onto the scaffold composition, wherein the scaffoldcomposition attracts a dendritic cell, introduces a immunogenic factorinto the dendritic cell thereby activating the dendritic cell, andinduces the dendritic cell to migrate away from the scaffoldcomposition. Alternatively the bioactive composition incorporated intoor coated onto the scaffold composition attracts a dendritic cell,introduces an immunogenic factor into the dendritic cell therebyactivating the dendritic cell, and induces the dendritic cell to migrateaway from the scaffold composition. In other preferred embodiments, thescaffold composition or bioactive composition separately attract adendritic cell to the device, introduce an immunogenic factor into thedendritic cell, and induce the dendritic cell to migrate away from thedevice.

DCs include conventional DCs as well as specific subsets of DCs. The TLRagonists, e.g., TLR3 agonists, preferentially attract and stimulateCD141+ DCs in the human (CD8+ DCs in the mouse). The TLR9 agonist, e.g.,CpG, preferentially attract and stimulate plasmacytoid DCs.

In preferred embodiments, the recruitment composition is GM-CSF, e.g.,encapsulated GM-CSF. The device temporally controls local GM-CSFconcentration, thereby controlling recruitment, residence, andsubsequent dispersement/deployment of immune cells to lymph nodes ortissue sites distant from location of the device, e.g., sites ofinfection or tumor location. The concentration of GM-CSF determineswhether if functions as a recruitment element or a deployment element.Accordingly, a method of programming dendritic cells in situ is carriedout by introducing to a subject a device comprising scaffold compositionand encapsulated recruitment composition. A pulse of recruitmentcomposition is released from the device within 1-7 days of introductionof the device, leaving a residual amount of the recruitment compositionin or on the device. The pulse is followed by slow release of theresidual amount over several weeks. The local concentration of therecruitment composition and the temporal pattern of release mediatesrecruitment, retention, and subsequent release of dendritic cells fromthe device. For example, the pulse comprises at least 50, 60, 75, 90 or95% of the amount of the recruitment composition associated with thedevice. An exemplary temporal release profile comprises a pulsecharacterized by release of at least 60% of the amount of therecruitment composition associated with the device in 1-5 days followingthe introduction of the device to a subject. Following the pulse, theresidual amount is slowly released over an extended period of time(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 days or 2, 3, 4, 5 or moreweeks) following the pulse period. Other recruitment compositionsinclude Flt3L and/or CCL20. The recruitment compounds are usedindividually or in combination.

The method of making a scaffold is carried out by providing a scaffoldcomposition, incorporating into or coating onto the scaffold compositiona first bioactive composition comprising polypeptides with means forattracting or repelling a dendritic cell, and contacting the scaffoldcomposition with a second bioactive composition, wherein the secondbioactive composition is covalently or non-covalently associated withthe scaffold composition wherein the second bioactive compositioncomprises a immunogenic factor. In an alternate embodiment of thismethod, the linking and contacting steps are repeated to yield aplurality of layers, wherein the second bioactive composition comprisesa combination of compounds with means to activate a dendritic cell.

Methods comprise continuous in situ dendritic cell programming,comprising administering to a subject, a device comprising a scaffoldcomposition and bioactive composition, the bioactive composition beingincorporated into or conjugated onto the scaffold composition, whereinthe scaffold composition attracts a dendritic cell, introduces aimmunogenic factor into the dendritic cell thereby activating thedendritic cell, and induces the dendritic cell to migrate away from thescaffold composition. The devices recruit and stimulate a heterogeneouspopulation of dendritic cells. Each subset is specialized andcontributes significantly to the generation of an immune response. Forexample, the device mediates CpG-ODN presentation and enrichment of asubset of dendritic cells, plasmacytoid DC (pDC), or CD141+ DCs, whichare particularly important in development of anti-tumor immunity.

Methods comprise increasing vaccine efficacy, comprising administeringto a subject, a device comprising a scaffold composition and bioactivecomposition, the bioactive composition being incorporated into orconjugated onto the scaffold composition, wherein the scaffoldcomposition attracts a dendritic cell, introduces a immunogenic factorinto the dendritic cell thereby activating the dendritic cell, andinduces the dendritic cell to migrate away from the scaffoldcomposition, thereby increasing the effectiveness of a vaccinationprocedure.

Methods comprise vaccinating a subject against cancer, comprisingadministering to a subject, a device comprising a scaffold compositionand bioactive composition, the bioactive composition being incorporatedinto or conjugated onto the scaffold composition, wherein the scaffoldcomposition attracts a dendritic cell, introduces a immunogenic factorinto the dendritic cell thereby activating the dendritic cell, andinduces the dendritic cell to migrate away from the scaffoldcomposition, thereby conferring upon a subject anti-tumor immunity,e.g., IL-12 production, and reduced tumor burden. In the case of alocalized or solid tumor, the device is administered or implanted at ornear the tumor site or site from which the tumor was excised orsurgically removed. For example, the device is implanted at a distanceof 1, 3, 5, 10, 15, 20, 25, 40 mm from a tumor site or site of excision,e.g., the PLG vaccine device is administered 16-21 mm away from a tumormass.

Immunogenic factors include TLR ligands. For example, the immunogenicfactor used is a modified TLR-9 ligand sequence, PEI-CpG-ODN.Preferably, the TLR ligand is a TLR3 agonist such as poly (I:C) orcondensed PEI-poly (I:C).

Scaffold compositions comprise a non-biodegradable material. Exemplarynon-biodegradable materials include, but are not limited to, metal,plastic polymer, or silk polymer. Moreover, scaffold compositions arecomposed of a biocompatible material. This biocompatible material isnon-toxic or non-immunogenic.

Bioactive compositions are covalently or non-covalently linked to thescaffold composition. Bioactive compositions comprise an element, eithercovalently or non-covalently bonded to the surface of the scaffoldcomposition, with means to attract a dendritic cell. Alternatively, orin addition, bioactive compositions comprise an element, eithercovalently or non-covalently bonded to the surface of the scaffoldcomposition, with means to introduce an immunogenic factor into adendritic cell. Alternatively, or further in addition, bioactivecompositions comprises an element, either covalently or non-covalentlybonded to the surface of the scaffold composition, with means to inducea dendritic cell to migrate away from the scaffold composition.

The element of the bioactive composition with means to manipulate adendritic cell is a secreted or membrane-bound amino acid, peptide,polypeptide, protein, nucleotide, dinucleotide, oligonucleotide,polynucleotide, polymer, small molecule or compound. In a preferredembodiment, this element is granulocyte macrophage colony stimulatingfactor (GM-CSF), because this element attracts dendritic cells to thescaffold composition. In another preferred embodiment, this element is aPEI-CpG-ODN sequence because this element has means to introduce CpG-ODNsequences into a dendritic cell thereby activating the cell. In someembodiments, this element is a polynucleotide or polypeptide encodingfor CCR7, a chemokine receptor that mediates dendritic cell migrationtowards lymph nodes and away from the scaffold composition. The CCR7element is introduced into a dendritic cell simultaneously orsequentially with PEI-CpG-ODN sequences to enhance dendritic cellmigration away from the scaffold composition.

Scaffold compositions of the present invention contain an externalsurface. Scaffold compositions of the present invention alternatively,or in addition, contain an internal surface. External or internalsurfaces of the scaffold compositions are solid or porous. Pore size isless than about 10 nm, in the range of about 100 nm-20 μm in diameter,or greater than about 20 μm. In preferred embodiments, the size of thepores allows the migration into and subsequent exit of cells such as DCsfrom the device. For example, the pores are nanoporous, microporous, ormacroporous. For example, the diameter of nanopores are less than about10 nm; micropore are in the range of about 100 μm-20 μm in diameter;and, macropores are greater than about 20 μm (preferably greater thanabout 100 μm and even more preferably greater than about 400 μm). In oneexample, the scaffold is macroporous with open, interconnected pores ofabout 100-500 μm in diameter, e.g., 100-200, 200-400, or 400-500 μm. Thesize of the pores and the interconnected architecture allows the cellsto enter, traverse within the volume of the device via theinterconnected pores, and then leave the device via the pores to go tolocations in the body outside of the device, e.g. to a tumor site, wherean immune response is mounted against tumor cells. The activated DCsmigrate away from the device and mount an immune response to solidtumors at discrete locations or throughout the body in the case ofmetastatic tumor cells or blood tumors such as leukemias.

Scaffold compositions of the present invention comprise one or morecompartments.

Devices of the present invention are administered or implanted orally,systemically, sub- or trans-cutaneously, as an arterial stent, orsurgically.

The devices and methods of the invention provide a solution to severalproblems associated with protocols for continuous cell programming insitu. In situ cell programming systems that stimulate immune responsesof the cells and induce their outward migration to populate infected ordiseased bodily tissues enhance the success of recovery, e.g., thespecific elimination of diseased tissue. Such a device that controlscell function and/or behavior, e.g., locomotion, contains a scaffoldcomposition and one or more bioactive compositions. The bioactivecomposition is incorporated into or coated onto the scaffoldcomposition. The scaffold composition and/or bioactive compositiontemporally and spatially (directionally) controls dendritic cellattraction, programming, and migration.

The devices mediate active recruitment, modification, and release ofhost cells from the material in vivo, thereby improving the function ofcells that have contacted the scaffold. For example, the device attractsor recruits cells already resident in the body to the scaffold material,and programs or reprograms the resident cells to a desired fate (e.g.,immune activation).

This device includes a scaffold composition which incorporates or iscoated with a bioactive composition; the device regulates attraction,activation, and migration of dendritic cells. Depending on theapplication for which the device is designed, the device regulatesattraction, activation, and/or migration of dendritic cells through thephysical or chemical characteristics of the scaffold itself. Forexample, the scaffold composition is differentially permeable, allowingcell migration only in certain physical areas of the scaffold. Thepermeability of the scaffold composition is regulated, for example, byselecting or engineering a material for greater or smaller pore size,density, polymer cross-linking, stiffness, toughness, ductility, orviscoelascticity. The scaffold composition contains physical channels orpaths through which cells can move more easily towards a targeted areaof egress of the device or of a compartment within the device. Thescaffold composition is optionally organized into compartments orlayers, each with a different permeability, so that the time requiredfor a cell to move through the device is precisely and predictablycontrolled. Migration is also regulated by the degradation, de- orre-hydration, oxygenation, chemical or pH alteration, or ongoingself-assembly of the scaffold composition.

Attraction, activation, and/or migration are regulated by a bioactivecomposition. The device controls and directs the activation andmigration of cells through its structure. Chemical affinities are usedto channel cells towards a specific area of egress. For example,cytokines are used to attract or retard the migration of cells. Byvarying the density and mixture of those bioactive substances, thedevice controls the timing of the migration. The density and mixture ofthese bioactive substances is controlled by initial doping levels orconcentration gradient of the substance, by embedding the bioactivesubstances in scaffold material with a known leaching rate, by releaseas the scaffold material degrades, by diffusion from an area ofconcentration, by interaction of precursor chemicals diffusing into anarea, or by production/excretion of compositions by resident supportcells. The physical or chemical structure of the scaffold also regulatesthe diffusion of bioactive agents through the device.

The bioactive composition includes one or more compounds that regulatecell function and/or behavior. The bioactive composition is covalentlylinked to the scaffold composition or non-covalently associated with thescaffold.

Signal transduction events that participate in the process of cellmigration are initiated in response to immune mediators. Thus, thedevice optionally contains a second bioactive composition that comprisesGM-CSF, a CpG-ODN or poly (I:C) sequence, a cancer antigen, and/or animmunomodulator.

In some cases, the second bioactive composition is covalently linked tothe scaffold composition, keeping the composition relatively immobilizedin or on the scaffold composition. In other cases, the second bioactivecomposition is noncovalently associated with the scaffold. Noncovalentbonds are generally one to three orders of magnitude weaker thancovalent bonds permitting diffusion of the factor out of the scaffoldand into surrounding tissues. Noncovalent bonds include electrostatic,hydrogen, van der Waals, π aromatic, and hydrophobic.

The scaffold composition is biocompatible. The composition isbio-degradable/erodable or resistant to breakdown in the body.Relatively permanent (degradation resistant) scaffold compositionsinclude metals and some polymers such as silk. Preferably, the scaffoldcomposition degrades at a predetermined rate based on a physicalparameter selected from the group consisting of temperature, pH,hydration status, and porosity, the cross-link density, type, andchemistry or the susceptibility of main chain linkages to degradation orit degrades at a predetermined rate based on a ratio of chemicalpolymers. For example, a high molecular weight polymer comprised ofsolely lactide degrades over a period of years, e.g., 1-2 years, while alow molecular weight polymer comprised of a 50:50 mixture of lactide andglycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks.A calcium cross-linked gels composed of high molecular weight, highguluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10,12 months) to years (1, 2, 5 years) in vivo, while a gel comprised oflow molecular weight alginate, and/or alginate that has been partiallyoxidized, will degrade in a matter of weeks.

Exemplary scaffold compositions include polylactic acid, polyglycolicacid, PLGA polymers, alginates and alginate derivatives, gelatin,collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, naturaland synthetic polysaccharides, polyamino acids, polypeptides,polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohols),poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modifiedstyrene polymers, pluronic polyols, polyoxamers, poly(uronic acids),poly(vinylpyrrolidone) and copolymers or graft copolymers of any of theabove. One preferred scaffold composition includes an RGD-modifiedalginate.

Another preferred scaffold composition a macroporouspoly-lactide-co-glycolide (PLG). For example, the PLG matrix includesGM-CSF, danger signals, and a target antigen, e.g., a cancer antigen andserves as a residence for recruited DCs as they are programmed. Therecruitment element, GM-CSF, is encapsulated into the PLG scaffolds. PLGmatrices that comprise the encapsulated GM-CSF provide a pulse of thedendritic cell recruitment composition and then a gradual slower rate ofrelease. The pulse comprises at least 40, 50, 60, 75, 80% or more of theinitial amount of bioactive composition with the remaining percent beingreleased gradually over the next days or weeks after administration tothe site in or on the subject to be treated. For example, release isapproximately 60% of bioactive GM-CSF load within the first 5 days,followed by slow and sustained release of bioactive GM-CSF over the next10 days. This release profile mediates a rate of diffusion of the factorthrough the surrounding tissue to effectively recruit resident DCs.

Porosity of the scaffold composition influences migration of the cellsthrough the device. Pores are nanoporous, microporous, or macroporous.For example, the diameter of nanopores are less than about 10 nm;micropore are in the range of about 100 nm-20 μm in diameter; and,macropores are greater than about 20 μm (preferably greater than about100 μm and even more preferably greater than about 400 μm). In oneexample, the scaffold is macroporous with aligned pores of about 400-500μm in diameter.

The device is manufactured in one stage in which one layer orcompartment is made and infused or coated with one or more bioactivecompositions. Exemplary bioactive compositions comprise polypeptides orpolynucleotides. Alternatively, the device is manufactured in two ormore (3, 4, 5, 6, . . . 10 or more) stages in which one layer orcompartment is made and infused or coated with one or more bioactivecompositions followed by the construction of a second, third, fourth ormore layers, which are in turn infused or coated with one or morebioactive compositions in sequence. Each layer or compartment isidentical to the others or distinguished from one another by the numberor mixture of bioactive compositions as well as distinct chemical,physical and biological properties.

A method of making a scaffold is carried out by providing a scaffoldcomposition and covalently linking or noncovalently associating thescaffold composition with a first bioactive composition. Exemplarydevices and methods of making them are described in U.S. Ser. No.12/867,426, Ser. No. 13/510,356, and PCT/US2012/35505, each of which ishereby incorporated by reference. The first bioactive compositionpreferably contains granulocyte macrophage colony stimulating factor.The scaffold composition is also contacted with a second bioactivecomposition, preferably one or more cytosine-guanosine oligonucleotide(CpG-ODN) sequences. The second bioactive composition is associated withthe scaffold composition to yield a doped scaffold, i.e., a scaffoldcomposition that includes one or more bioactive substances. Thecontacting steps are optionally repeated to yield a plurality of dopedscaffolds, e.g., each of the contacting steps is characterized by adifferent amount of the second bioactive composition to yield a gradientof the second bioactive composition in the scaffold device. Rather thanaltering the amount of composition, subsequent contacting steps involvea different bioactive composition, i.e., a third, fourth, fifth, sixth .. . , composition or mixture of compositions, that is distinguished fromthe prior compositions or mixtures of prior doping steps by thestructure or chemical formula of the factor(s). The method optionallyinvolves adhering individual niches, layers, or components to oneanother and/or insertion of semi-permeable, permeable, or nonpermeablemembranes within or at one or more boundaries of the device to furthercontrol/regulate locomotion of cells or bioactive compositions.

Therapeutic applications of the device include the instruction of immunecells. For example, the method includes the steps of providing a devicethat includes scaffold composition with a bioactive compositionincorporated therein or thereon and a mammalian cell bound to thescaffold and contacting a mammalian tissue with the device, e.g., byimplanting or affixing the device into or onto a mammalian tissue. Atthe time of administering or implanting the device, exemplary relativeamounts of each component, recruiting composition (e.g., GM-CSF, Flt3L,or CCL20), danger signal (e.g., CpG-ODN), and antigen (e.g., purifiedtumor antigen or tumor cell lysate) are as follows: GM-CSF: 0.5 μg-500μg; CpG-ODN: 50 μg-3,000 μg; and Tumor antigen/lysate: 100 μg-10,000 μg.

A method of modulating an activity of a cell, e.g., a host cell, iscarried out by administering to a mammal a device containing a scaffoldcomposition and a recruitment composition incorporated therein orthereon, and then contacting the cell with a deployment signal. Thecells leave the device after encountering antigen (and other factors)and thus being activated to seek out tumor cells in the body to which animmune response is mounted. The activity of the cell at egress differsfrom that prior to entering the device. Cells are recruited into thedevice and remain resident in the device for a period of time, e.g.,minutes; 0.2, 0.5, 1, 2, 4, 6, 12, 24 hours; 2, 4, 6, days; weeks (1-4),months (2, 4, 6, 8, 10, 12) or years, during which the cells are exposedto structural elements and bioactive compositions that lead to a changein the activity or level of activity of the cells. Encountering theantigen and other compounds in the device induces egress of the altered(re-educated or reprogrammed) cells, and the cells migrate out of thedevice and into surrounding tissues or remote target locations to seekout and mediate immunity against diseased cells such as tumor cells.

The deployment signal is a composition such as protein, peptide, ornucleic acid or a state of activation of the cell. For example, havingingested antigen, DCs become activated and migrate to lymph nodes, thespleen, and other anatomical locations, where they meet up with T cellsto further propagate an antigen-specific immune response, e.g.,anti-cancer response. For example, cells migrating into the device onlyencounter the deployment signal once they have entered the device. Insome cases, the deployment signal is a nucleic acid molecule, e.g., aplasmid containing sequence encoding a protein that induces migration ofthe cell out of the device and into surrounding tissues. The deploymentsignal occurs when the cell encounters the plasmid in the device, theDNA becomes internalized in the cell (i.e., the cell is transfected),and the cell manufactures the gene product encoded by the DNA. In somecases, the molecule that signals deployment is an element of the deviceand is released from the device in delayed manner (e.g., temporally orspatially) relative to exposure of the cell to the recruitmentcomposition. Alternatively, the deployment signal is a reduction in orabsence of the recruitment composition. For example, a recruitmentcomposition induces migration of cells into the device, and a reductionin the concentration or depletion, dissipation, or diffusion of therecruitment composition from the device results in egress of cells outof the device. In this manner, immune cells such as T cells, B cells, ordendritic cells (DCs) of an individual are recruited into the device,primed and activated to mount an immune response against anantigen-specific target. Optionally, an antigen corresponding to atarget to which an immune response is desired is incorporated into oronto the scaffold structure. Cytokines, such as granulocyte macrophagecolony stimulating factor (GM-CSF) are also a component of the device toamplify immune activation and/or induce migration of the primed cells tolymph nodes. Other cell specific recruitment compositions are describedbelow.

The device recruit cells in vivo, modifies these cells, and thenpromotes their migration to another site in the body. This approach isexemplified herein in the context of dendritic cells and cancer vaccinedevelopment but is also useful to other vaccines such as those againstmicrobial pathogens as well as cell therapies in general. Cells educatedusing the devices described herein promote regeneration of a tissue ororgan immediately adjacent to the material, or at some distant site.Alternatively, the cells are educated to promote destruction of a tissue(locally or at a distant site). The methods are also useful for diseaseprevention, e.g., to promote cell-based maintenance of tissue structureand function to stop or retard disease progression or age-related tissuechanges. The education of cells within the device, “programming” and“reprogramming” permits modification of the function or activity of anycell in the body to become a multipotent stem cell again and exerttherapeutic effects.

The inability of traditional and ex vivo DC-based vaccination strategiesto coordinate and sustain an immune response mediated by theheterogeneous DC network in cancer patients has led to limited clinicaleffectiveness of these approaches. The devices and methods describedherein have distinct advantages, because preferential recruitment andexpansion of pDCs dramatically improves immune responses to cancerantigens and reduces tumor progression compared to previous vaccineapproaches.

Polynucleotides, polypeptides, or other agents are purified and/orisolated. Specifically, as used herein, an “isolated” or “purified”nucleic acid molecule, polynucleotide, polypeptide, or protein, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Purified compounds are at least60% by weight (dry weight) the compound of interest. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Forexample, a purified compound is one that is at least 90%, 91%, 92%, 93%,94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight.Purity is measured by any appropriate standard method, for example, bycolumn chromatography, thin layer chromatography, or high-performanceliquid chromatography (HPLC) analysis. A purified or isolatedpolynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA))is free of the genes or sequences that flank it in itsnaturally-occurring state. A purified or isolated polypeptide is free ofthe amino acids or sequences that flank it in its naturally-occurringstate. Purified also defines a degree of sterility that is safe foradministration to a human subject, e.g., lacking infectious or toxicagents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones. Forexample, the isolated nucleic acid is a purified cDNA or RNApolynucleotide. Isolated nucleic acid molecules also include messengerribonucleic acid (mRNA) molecules.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the immune response to infection. FIG. 1 is adiagram showing the mechanisms by which bacterial invasion and bacterialtoxins damage resident skin cells promoting the production ofinflammatory cytokines, including GM-CSF, and activation of dermalendothelium. Cytokine stimulation induces extravasation of leukocytesand recruits skin resident DCs (langerhans cells) and monocytes/preDCs.DCs, recruited to the site of inflammation encounter and ingestbacterium and bacterial products including antigenic molecules andCpG-rich DNA, which stimulates TLR9 activation. As a result of TLRligation and the inflammatory conditions, the DC rapidly matures toupregulate its expression of MHC-antigen complexes, costimulatorymolecules, and CCR7 and begins to home to the lymph nodes where itinitiates and propagates antigen specific T-cell responses.

FIGS. 2A-C. FIG. 2A is a schematic representation of PEI condensation ofCpG-rich oligonucleotide sequences. The PEI polycation with positivelycharged amine groups is mixed with CpG-ODNs consisting of negativelycharged phosphate groups at charge ratios (NH3+:PO4−) resulting inpositively charged PEI-CpG-ODN condensates. FIG. 2B is a bar graphshowing the zeta potential (my) of CpG-ODN 1826 and its PEI condensatesat charge ratios of 4, 7 and 15. Box plots represent the mean andstandard deviation (n=4) FIG. 2C is a bar graph showing the particlesize of CpG-ODN 1826 and its PEI condensates at charge ratios of 4, 7and 15. Values represent the average particle size and the standarddeviation (n=4).

FIGS. 3A-D. FIGS. 3A-C show in vitro uptake of CpG-ODN by JAWSII DCs.

FIGS. 3A-B are bright field images of cells and their correspondingfluorescent images displaying the uptake of TAMRA labeled CpG-ODNmolecules (A) or PEI-CpG-ODN condensates (B). FIG. 3C is a bar graphshowing quantification of uptake of naked (-o-) and PEI-CpG-ODN (-▪-)condensates over a period of 110 hours. FIG. 3D is a line graph showingquantification of uptake of PEI-CpG-ODN condensates and subsequentdecondensation within JAWSII DCs. The number of PEI-CpG-ODN condensatesin the cells (-▪-), and the amount of uncondensed CpG-ODN (--□--) wasmonitored and quantified over a period of 70 hours. Scale bar—20 μm.Values in C (n>10 cells) and D (n>7 cells) represent the mean andstandard deviation.

FIGS. 4A-D. (A) Imaging DC activation. FIG. 4A is a series ofbrightfield images of activated DC morphology in correlation withfluorescent images displaying the uptake of TAMRA labeled CpG-ODNmolecules condensed with PEI (charge ratio −7). FIG. 4B is a series ofFACS histograms of JawsII DCs positive for the activation markers CD86,MHCII and CCR7 following no stimulation (tinted line), CpG-ODN (- - -),and PEI-CpG-ODN condensates (-). FIG. 4C is a chart showing tabulateddata displaying the percentage of DCs positive for the activationmarkers CD86, MHCII, and CCR7 following no stimulation, and stimulationwith TNF-α/LPS or CpG-ODN or PEI-CpG-ODN. FIG. 4D is a bar graph showingCpG-ODN and DC emigration toward CCL19. The effects of no stimulation(▪), and PEI (▪) or CpG-ODN (▪) or PEI-CpG-ODN (▪) stimulation on DCemigration from the top wells of transwell systems toward mediasupplemented with 300 ng/ml CCL19. Migration counts taken at 24 hours.Scale bar—20 μm. Values in C and D (n=4) represent the mean and standarddeviation. CpG-ODN activation media (5 μg/ml). * P<0.05** P<0.01.

FIGS. 5A-B. FIG. 5A is a series of bar graphs showing the percentage ofJawsII DCs positive for MHCII and CCR7 expression after PEI-CpG-ODN (5μg/ml) stimulation in media supplemented with 0 (□), 50 (▪) and 500ng/ml GM-CSF (▪). FIG. 5B is a line graph showing CpG-ODN and DCemigration toward CCL19 in the presence of GM-CSF. The effects of nostimulation (-▪-), and stimulation with PEI (---) or CpG-ODN (--) orPEI-CpG-ODN (--) on DC emigration from the top wells of transwellsystems toward media supplemented with 300 ng/ml CCL19. Migration countstaken at 24 hours. Values represent the mean and standard deviation(n=4).

FIGS. 6A-C. FIG. 6A is a line graph showing the fraction of PEI-CpG-ODNcondensates retained in PLG matrices over time with incubation in PBS invitro. FIGS. 6B-C are bar graphs showing emigration of JAWS II DCs fromCpG-ODN loaded scaffolds. (B) The total number of DCs that migrated fromscaffolds loaded with 5, 50, 500 μg of CpG-ODN toward media supplementedwith 300 ng/ml CCL19. (C) The total number of DCs that migrated fromscaffolds loaded with 25 μg of CpG-ODN in the presence of 500 ng/mlGM-CSF toward media supplemented with 300 ng/ml CCL19. Migration countstaken at 48 hours. Values represent mean and standard deviation (n=4 or5).

FIGS. 7A-B. PLG-based infection mimics continuously program DCs in situ.FIG. 7A is a chart showing the tabulated data of host DC recruitment(cell #) and DC activation (% expressing MHC or CCR7) in response tovarious dosages of PEI-CpG-ODN and GM-CSF loaded into PLG matrices.Matrices were implanted into the backs of C57/BL6J mice for 7 days. FIG.7B is a bar graph showing the number of CD11c(+)MHCII(+) andCD11c(+)CCR7(+) host DCs isolated from matrices loaded with PEI-ODNcontrol, 10 μg PEI-CpG-ODN, 400 and 3000 ng GM-CSF, and 400 and 3000 ngGM-CSF in combination with 10 μg PEI-CpG-ODN at Day 7 after implantationinto the backs of C57/BL6J mice. Values represent the mean and standarddeviation (n=3-5). * P<0.05** P<0.01.

FIGS. 8A-D. Infection mimics continuously disperse programmed DCs insitu. FIG. 8A is a bar graph showing the number of FITC(+) DCs that havehomed to the inguinal lymph nodes as a function of time subsequent totheir residence at FITC painted blank matrices (-□-), FITC paintedGM-CSF loaded matrices (--), and FITC painted GM-CSF and CpG-ODNmatrices (--). GM-CSF dose was 3000 ng and CPG-ODN dose was 10 μg. FIG.8B is a digital photograph of inguinal lymph nodes extracted fromC57BL/6J mice (control) and at 10 days after the implantation ofmatrices incorporating 10 μg CpG-ODN+3000 ng GM-CSF (infection-mimic)FIGS. 8C-D are bar graphs showing the total number of cells (C) andCD11c+ DCs (D) isolated from inguinal lymph nodes extracted fromC57BL/6J mice at 2 and 7 days after the implantation of blank matrices(□) and matrices incorporating 3000 ng GM-CSF (▪) or 10 μg CpG-ODN+3000ng GM-CSF (▪). Values in A, C and D represent the mean and standarddeviation (n=4 or 5). * P<0.05** P<0.01.

FIG. 9 is a bar graph showing infection-mimicking microenvironmentconfers potent anti-tumor immunity. The time to tumor occurrence afterPLG cancer vaccines were implanted into mice. A comparison between blankPLG scaffolds (Blank), scaffolds loaded with antigen alone (Lys),antigen+3000 ng GM-CSF (Lys+3000 ng GMCSF), antigen+ PEI-CpG-ODNcondensate (Lys+CpG) and the combination of antigen, 3000 ng GM-CSF andPEI-CpG-ODN (Lys+3000 ng+ PEI-CpG-ODN). Animals were also immunizedusing a cell-based vaccine (cell-based) using irradiated B16-F10melanoma cells that had been genetically modified to produce GM-CSF, forcomparison. At Day 14 after vaccination, C57BL/6J mice were challengedwith 10⁵ B16-F10 melanoma tumor cells and monitored for the onset oftumor occurrence (n=9 or 10).

FIGS. 10A-B. Vaccination efficacy of Infection mimics dependent on Tcell responses. FIG. 10A is a series of representative photomicrographsof tumor sections from mice vaccinated with PLG cancer vaccines thatappropriately control the presentation of tumor lysates, 3000 ng GM-CSFand CpG-ODN and blank (blank) scaffold controls. Sections were stainedto detect for CD4(+) and CD8(+) T cell infiltrates into tumor tissuethat was explanted from mice that had developed tumors at days 20-25.FIG. 10B is a bar graph showing T-cell infiltrates into B16-FI0 melanomatumors of vaccinated animals. Tumors were explanted from C57BL/6J micetreated with blank PLG scaffolds (▪), or PLG scaffolds incorporatingB16-F10 melanoma tumor lysates, 3000 ng GM-CSF and 10 μg PEI-CpG-ODN (▪)at days 20-25. T-cell infiltrates were examined in randomized sectionsof tumors (n=4, 1 mm³) Scale bar—50 μm. Values in A, D and E representthe mean and standard deviation (n=3 or 4). * P<0.05** P<0.01.

FIGS. 11 A-F. In vivo control of DC recruitment and programming. FIG.11A is a line graph showing cumulative release of GM-CSF from PLGmatrices over a period of 23 days. FIG. 11B is a photograph showing H&Estaining of sectioned PLG scaffolds explanted from subcutaneous pocketsin the backs of C57BL/6J mice after 14 days: Blank scaffolds, and GM-CSF(3000 ng) loaded scaffolds. FIG. 11C is a series of FACS plots of cellsisolated from explanted scaffolds and stained for the DC markers, CD11cand CD86. Cells were isolated from blank and GM-CSF (3000 ng) loadedscaffolds implanted for 28 days. Numbers in FACS plots indicate thepercentage of the cell population positive for both markers. FIG. 11D isa bar graph showing the fractional increase in CD11c(+)CD86(+) DCsisolated from PLG scaffolds at day 14 after implantation in response todoses of 1000, 3000 and 7000 ng of GM-CSF, as normalized to the blankcontrol (Blanks). FIG. 11E is a line graph showing the in vivoconcentration profiles of GM-CSF at the implant site of PLG scaffoldsincorporating 0 (−), 3000 (--◯--), and 7000 ng (--▪--) of GM-CSF as afunction of time post implantation. FIG. 11F is a bar graph showing thepercentage of CD11c(+)CCR7(+) host DCs isolated from scaffolds loadedwith 0 (▪), 400 (▪), 3000 ng (▪), and 7000 ng of GM-CSF (©) as afunction of time after implantation into the backs of C57BL/6J mice.Scale bar in B—500 μm. Values in A, D, E, and F represent mean andstandard deviation (n=4 or 5). * P<0.05 ** P<0.01.

FIGS. 12 A-G. Antigen co-presentation with CpG-ODN to DCs infiltratingPLG matrices enhances local CD8+cDC numbers, IL-12 production and totalCD8(+) cell numbers. The number of (FIG. 12A) plasmacytoid DCs, (B)CD11c(+)CD11b(+) cDCs, and (FIG. 12C) CD11c(+)CD8(+) cDCs at day 10post-implantation in blank matrices (Blanks) and in response to doses of3000 ng GM-CSF (GM) or 100 μg CpG-ODN (CpG) alone or in combination(CpG+GM) or co-presented with tumor lysates (GM+Ant, CpG+Ant andCpG+GM+Ant). The in vivo concentration of (FIG. 12D) IFN-α (E) IFN-γ and(FIG. 12F) IL-12 at day 10 post-implantation in blank matrices (Blanks)and in response to doses of 3000 ng GM-CSF (GM) or 100 μg CpG-ODN (CpG)alone or in combination (CpG+GM) or co-presented with tumor lysates(GM+Ant, CpG+Ant and CpG+GM+Ant). (FIG. 12G). FACS histograms of CD8(+)cells infiltrating Blank PLG matrices(-), and matrices loaded with 3000ng GM-CSF and 100 μg CpG-ODN alone (- - -) or with tumor antigens(tinted line). Values in A-F represent mean and standard deviation (n=4or 5). * P<0.05 ** P<0.01.

FIGS. 13A-F. Tumor protection regulated by CpG-ODN presentation andplasmacytoid DC enrichment. Survival times of mice vaccinated with PLGvaccines 14 days prior to B16-F10 melanoma tumor challenge. (FIG. 13A)shows a comparison of survival times in mice vaccinated with PLGmatrices loaded with tumor lysates and 1, 10, 50 or 100 μg of CpG-ODN.FIG. 13B shows a comparison of survival times in mice vaccinated withPLG matrices loaded with tumor lysates, 3000 ng GM-CSF and 1, 10, 50 or100 μg of CpG-ODN. A correlation between the number of (FIG. 13C)CD11c(+)PDCA-1(+) DCs, (FIG. 13D) CD11c(+)CD11b(+) DCs, and (FIG. 13E)CD11c(+)CD8(+) cDCs at the PLG vaccine site at day 10 and the percent ofanimals surviving B16-F10 melanoma tumor challenge at Day 100. FIG. 13Fshows the fraction of total DC population consisting of CD11c(+)CD11b(+)cDCs, CD11c(+)PDCA-1(+) pDCs, and CD11c(+)CD8(+) cDCs generated at thePLG vaccine site at day 10. Survival percentage is taken at Day 100after challenge with B16-F10 melanoma cells.

FIGS. 14A-B are line graphs showing PLG vaccine efficacy againstestablished tumors. FIG. 14A shows a comparison of the survival time inC57BL/6 mice treated with blank PLG scaffolds, and PLG vaccines (3 μgGM-CSF+100 μg CpG-ODN+tumor lysates). FIG. 14B shows a comparison oftumor growth in C57BL/6 mice treated with blank PLG scaffolds, and PLGvaccines (3 μg GM-CSF+100 μg CpG-ODN+tumor lysates). Mice wereinoculated with 5×10⁵ B16-F10 melanoma tumor cells at Day 0 and tumorswere allowed to grow for 7 days when mice were either implanted withblank PLG matrices or PLG vaccine. The average tumor size was expressedas one-half the product of the smallest and largest diameter.

FIGS. 15A-B are line graphs showing cumulative release of (A) CpG-richoligonucleotides (CpG 1826) or (B) P(I:C) in combination with thecumulative release of GM-CSF from PLG scaffolds. FIGS. 15C-D are bargraphs showing bioactivity of P(I:C) and MPLA, respectively, releasedfrom PLG scaffolds presented as fold-increase over controls. Bioactivitymeasured by the ability of the Poly(I:C) and MPLA released from PLGscaffolds to stimulate HEK293 cells expressing TLR3 and TLR4,respectively, and stably transfected with NF-κB-dependent alkalinephosphatase reporter. Bioactivity was measured over time and compared tocontrols of unstimulated cells. FIG. 15E is a line graph showing MPLA incombination with the cumulative release of GM-CSF from PLG scaffolds.Values represent mean and standard deviation (n=5 or 6). These data showin vitro release kinetics and bioactivity of various TLR agonists fromGM-CSF loaded PLG scaffolds. FIG. 15F is a photomicrograph showing thetop surface of a macroporous PLG scaffold (scale bar—3 mm), and SEMmicrograph of a scaffold cross section (scale bar—50 μm).

FIGS. 16A-D are bar graphs showing DC recruitment and Activation atVaccine Site is regulated by TLR agonist presentation. The total numbersof (A) CD11c(+) DCs, (B) activated CD11c(+) DCs positive for MHCII andCD86 expression, (C) PDCA-1(+) plasmacytoid DCs, and (D) CD11c(+)CD8(+)DCs recruited to scaffold site at day 7 after implantation of GM-CSFloaded matrices (GM) and matrices loaded with GM-CSF in combination withCpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)). FIG. 16E shows FACShistograms and plots representing scaffold infiltrating dendritic cellsin GM-CSF loaded scaffolds (Con) or scaffolds loaded with GM-CSF incombination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)) at day 7postimplantation in mice. Histograms indicate the relative frequency ofCD11c(+) dendritic cells infiltrating the indicated scaffoldformulation. Density plots indicate cells stained for CD11c(+) incombination with activated, DC markers, CD86(+) and MHCII(+). Numbers inthe upper right quadrant of FACS plots indicate the percentage ofCD11c(+) dendritic cells positive for activation markers. FIG. 16F is abar graph showing the total numbers of CD11c(+) DCs, and activatedCD11c(+) DCs positive for MHCII and CD86 expression, at the scaffoldsite at day 7 after implantation of GM-CSF loaded matrices (Con) andmatrices loaded with GM-CSF in combination with CpG-ODN (CpG), MPLA(MPLA), and P(I:C) (P(IC)). Values represent mean and standarddeviations (n=6). * P<0.05 ** P<0.01, as compared to GM-CSF loadedmatrices. ** P<0.01, as compared to GM-CSF loaded matrices (Con).

FIGS. 16G and 16H are bar charts showing that CD8(+)DC, and pDC subsetsand IL-12 concentrations at vaccine site. FIG. 16G shows the totalnumbers of CD11c(+)CD8(+) DCs and pDCs at scaffold site at day 7, and(H) the local IL-12 concentration after implantation of GM-CSF loadedscaffolds (Con) and scaffolds loaded with GM-CSF in combination withCpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)). Values represent meanand standard deviations (n=6). ** P<0.01, as compared to GM-CSF loadedmatrices (Con).

FIGS. 17A-D are graphs showing prophylactic vaccination and correlationto CD8(+)DC, and pDC subsets and IL-12 concentrations at vaccine site.Survival times of mice vaccinated with PLG vaccines 14 days prior toB16-F10 melanoma tumor challenge (10⁵ cells). FIG. 17A shows acomparison of survival times in untreated mice (Control) and micetreated with GMCSF loaded PLG scaffolds (GM-CSF) or with PLG scaffoldsloaded with GM-CSF in combination with CpG-ODN (CpG), P(I:C), or MPLA.Plots of the normalized magnitude of (B) CD11c(+)CD8(+) DC infiltration,(C) pDC infiltration at the vaccine site, and (D) local IL-12concentration versus the percent of animals surviving B16-F10 melanomatumor challenge at Day 100 (survival data taken from experimentalconditions in (A; red data points) and previously reported data withthis system). r values in B-C represent the linear correlationcoefficient between x-axis variable and survival percentage.

FIGS. 18A-D are bar graphs showing T cell Activity and cytokineproduction at vaccine site at Day 14 of scaffold implantation. (A) Thenumber of CD3(+)CD8(+) cytotoxic T cells at day 14 after implantation ofGM-CSF loaded matrices (GM) and matrices loaded with GM-CSF incombination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)). The invivo concentration of (B) IL-12, (C) Rantes, and (D) IFN-g at day 14after implantation of GM-CSF loaded matrices (GM) and matrices loadedwith GM-CSF in combination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C)(P(IC)). Values represent mean and standard deviations (n=5). * P<0.05** P<0.01, as compared to control matrices (loaded with GM-CSF).

FIGS. 19A-F are graphs showing therapeutic vaccination and anti-tumor Tcell activity. A comparison of the (A) tumor size and (B) overallsurvival in mice bearing established melanoma tumors (inoculated with5×10⁵ B16-F10 cells and allowed to develop for 9 days) and treated witheither GM-CSF loaded matrices (Con) or matrices loaded with GM-CSF incombination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)). (C)FACS plots representing tumor infiltrating leukocytes isolated fromexplanted tumors at Day 18 after tumor challenge. Mice were treated withGM-CSF loaded matrices (Control) or matrices loaded with GM-CSF incombination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)) at Day 9after tumor inoculation and cell isolations from tumors were prepared atDay 18 and stained for activated, cytotoxic T cell markers, CD8(+) andCD107a. Numbers in FACS plots indicate the percentage of the cellpopulation positive for both markers. (D) The numbers of CD8(+),tumor-infiltrating T cells positive for both IFNγ and CD107a inuntreated mice (naïve) or mice vaccinated with various treatments. (E)The total numbers of Trp2-specific cytotoxic T cells in splenocytes ofvaccinated mice. FIG. 19F is a bar chart showing a comparison of thetumor size at Day 17. * P<0.05 ** P<0.01, as compared to controlmatrices (loaded with GM-CSF), unless otherwise noted.

FIGS. 20A-D are a series of bar charts, line graphs and dot plotsshowing that vaccine efficacy is impaired in mice lacking CD8(+) DC. (A)Survival times of untreated mice (control), wildtype C57BL/6J mice (WT)and Batf3−/− mice (CD8 DC KO), vaccinated with PLG vaccines 14 daysprior to B16-F10 melanoma tumor challenge (105 cells). (B) Analysis ofcytotoxic and regulatory T cells at PLG vaccine site of wildtypeC57BL/6J mice (WT) and Batf3−/− mice (CD8 DC KO) at day 10 postimplantation. FACS dot plots indicate scaffold-infiltrating cellsstained for CD3(+) and Trp2(+) tetramer. Numbers in the upper rightquadrant of FACS plots indicate the percentage Trp2 specific cytotoxic Tcell, and numbers in lower right quadrant represent the rest of the Tcell population at the vaccine site. Graphs indicate the total numbersof Trp2-specific cytotoxic T cells at implant site and the ratio ofCD8(+) cytotoxic T cells to regulatory T cells. (C) Fold increase inIL-12 concentration at vaccine site and (D) Trp(2)-specific cytotoxic Tcells in spleens of vaccinated, wildtype C57BL/6J mice (WT) and Batf3−/−mice (CD8 DC KO). CpG-ODN was the adjuvant utilized in vaccines. Datarepresent mean and standard deviation, (n=5)* P<0.05 ** P<0.01.

FIGS. 21A-D are a series of bar charts. FIG. 21A shows the local TNF-αconcentration after implantation of GM-CSF loaded matrices (Con) andscaffolds loaded with GM-CSF in combination with CpG-ODN (CpG), MPLA(MPLA), and P(I:C) (P(IC)). Values represent mean and standarddeviations (n=5). ** P<0.01, as compared to GM-CSF loaded matrices(Con). FIG. 21B shows the local IFN-α concentration after implantationof GM-CSF loaded scaffolds (Con) and scaffolds loaded with GM-CSF incombination with CpG-ODN (CpG), MPLA (MPLA), and P(I:C) (P(IC)). Valuesrepresent mean and standard deviations (n=5). **P<0.01, as compared toGM-CSF loaded matrices (Con). FIG. 21C shows the local IL-12p70concentration after implantation of GM-CSF loaded scaffolds (Con) andvaccine scaffolds (loaded with tumor lysate, GM-CSF and CpG-ODN)(CpG),in wildtype (vax) and Cd8atm1Mak/J mice (CD8 Tc KO). FIG. 21D shows thelocal IFN-γ concentration after implantation of GM-CSF loaded scaffolds(Con) and vaccine scaffolds (loaded with tumor lysate, GM-CSF andCpG-ODN)(CpG), in wildtype (vax) and B6.12952-Cd8atm1Maka mice (CD8 TcKO).

FIG. 22 is a line graph showing that PLG vaccines incorporating CpG-ODNand/or P(I:C) generate significant tumor protection. The overallsurvival of mice bearing melanoma tumors, and treated with either blankmatrices [Blank] or matrices loaded with CpG-ODN or P(I:C) alone or incombination [CpG-ODN+P(I:C)] (n=8). Mice were challenged with 5×10⁵B16-F10 cells and vaccinated 3 days later with PLG vaccines. Total doseof TLR agonist was approximately 100 μg in all vaccines.

FIGS. 23A-B are bar charts demonstrating in vitro chemotaxis andchemokinesis of DCs. The in vitro (FIG. 23A) chemotaxis and (FIG. 23B)chemokinesis of bone marrow derived DCs in response to control media andmedia supplemented with GMCSF, Flt3L, and CCL20. * P<0.05 ** P<0.01, ascompared to GM-CSF loaded matrices. Values represent mean and standarddeviation. (n=4).

FIGS. 24A-C are a series of line graphs, bar charts, andphotomicrographs demonstrating PLG scaffolds that release cytokines forDC recruitment. FIG. 24A shows the cumulative release of GM-CSF, Flt3L,or CCL20 from PLG scaffolds. FIG. 24B shows a representative photographof scaffold histological sections stained for CD11(+) DC infiltrates(pink) into macroporous blank (left) and GM-CSF loaded scaffolds (right)at Day 10 after implantation. Scale bar—100 μm. FIG. 24C shows the totalnumbers of CD11c(+) DCs at scaffold site at day 7 after implantation ofBlank PLG matrices (Con) and matrices loaded with GM-CSF (GM), Flt3L(FL3) and CCL20 (CCL20). Values represent mean and standard deviations(n=6). * P<0.05 ** P<0.01, as compared to GM-CSF loaded matrices.

FIGS. 25A-C are a series of line graphs, dot plots, and bar chartsdemonstrating DC recruitment and activation mediated by PLG matricesloaded with Cytokines and CpG-ODN. FIG. 25A shows FACS histograms andplots representing scaffold infiltrating dendritic cells in CpG-ODNloaded PLG scaffolds (Con) or scaffolds loaded with GM-CSF (GM),Flt3L(F13L) or CCL20(CCL20) in combination with CpG-ODN at day 7post-implantation in mice. Histograms indicate the relative frequency ofMHCII and CD86 expression in CD11c(+) DCs infiltrating the indicatedscaffold formulation. Dot plots indicate cells stained for CD11c(+) incombination with activated, plasmacytoid DC marker, PDCA-1. Numbers inthe upper right quadrant of FACS plots indicate the percentage ofCD11c(+)PDCA-1(+) pDCs. FIG. 25B shows the total numbers of activatedCD11c(+) DCs positive for MHCII and CD86 expression, and FIG. 25C showsCD11c(+)PDCA-1(+) pDCs present in scaffold at day 7 after implantationof CpG-ODN loaded PLG scaffolds (Con) or scaffolds loaded with GM-CSF(GM), Flt3L(F13L) or CCL20(CCL20) in combination with CpG-ODN. Valuesrepresent mean and standard deviations (n=5). * P<0.05 ** P<0.01, ascompared to controls (Con) unless otherwise indicated.

FIGS. 26A-D are a series of bar charts and a line graph demonstratingPLG vaccines generate immunoprotective cytokines, antigen-specific Tcells, and cancer protection. Fold difference in local (FIG. 26A) IL-12and (FIG. 26B) IFN-γ concentration after implantation of scaffoldsloaded with B16-F10 tumor lysate, CpG-ODN in combination with GM-CSF(GM), Flt3L(F13L) or CCL20(CCL20). Concentrations were normalized to thevalue found with control (matrices delivering lysate and CpG-ODN, nocytokines) (FIG. 26C). The total numbers of Trp2-specific CD8(+) T cellsin spleens of vaccinated animals at Day 10 post-implantation. (FIG. 26Dshows the overall survival of mice bearing melanoma tumors, and treatedwith either CpG-ODN loaded matrices (Blank) or matrices loaded withCpG-ODN in combination with GMCSF, Flt3L and CCL20 (n=8). Valuesrepresent mean and standard deviations (n=5). * P<0.05, as compared toCCL20 loaded matrices (CC20).

DETAILED DESCRIPTION OF THE INVENTION

Prior to the invention, cancer vaccines typically depended on cumbersomeand expensive manipulation of cells in the laboratory, and subsequentcell transplantation resulted in poor lymph node homing and limitedefficacy. The invention solves these problems by using materials thatmimic key aspects of bacterial infection to directly control immune celltrafficking and activation in the body. Presentation of TLR agonists forcancer vaccination leads to improved activation of immune cells. Thevaccines and methods comprise incorporation and presentation of TLRagonists embedded in structural polymeric devices. The data describedherein demonstrate the critical role of CD8(+) Dendritic cells (DCs) andplasmacytoid DCs (as well as conventional DCs) for cancer vaccination,which are preferentially recruited and activated using the TLR-agonistcontaining structural polymeric device. The device is manufactured as atiny bioengineered porous disc filled with tumor-specific antigens andTLR agonists. The disc is implanted into the body, e.g., inserted underthe skin, where it activates the immune system to destroy tumor cells.While typical earlier methods involved growing cells outside the body,this approach reprograms cells that are already in the body.

In some examples, the device includes a recruitment component. Thus, thedevice optionally includes a recruitment molecule such as a cytokine. Inthose situations, polymers were designed to first release a cytokine torecruit and house host dendritic cells (DCs), and subsequently presentcancer antigens and danger signals to activate the resident DCs anddramatically enhance their homing to lymph nodes. Specific andprotective anti-tumor immunity was generated with these materials, as90% survival was achieved in animals that otherwise die from cancerwithin 25 days. These materials are useful in cancer and other vaccinesto program and control the trafficking of a variety of cell types in thebody.

A polymer system was designed to not only serve as a drug deliverydevice, but also as a physical, antigen-presenting structure to whichthe DCs are recruited, and where DCs reside while they are activatedusing a material (poly[lactide-co-glycolide]) and bioactive molecules(GM-CSF and CpG-ODN). These bioactive molecules have excellent safetyprofiles. The material system serves as an effective cancer vaccine,eliminating the time, expense and regulatory burden inherent to existingcell therapies and reducing or eliminating the need for multiple,systemic injections and high total drug loading. The devices describedherein utilize infection-mimicking materials to program DCs in situ.

A quantitative understanding of the ability of GM-CSF to impact DCrecruitment, activation and emigration in vitro was developed in orderto appropriately design a material system for vaccination. GM-CSFenhanced DC recruitment and proliferation in a dose dependent mannerHowever, high concentrations (>100 ng/ml) of GM-CSF inhibited DCmigration toward a lymph node derived chemoattractant (CCL19)Immunohistochemical staining revealed that the high concentrations ofGM-CSF (500 ng/ml) also down-regulated DC expression of the CCL19receptor CCR7 and MHCII. These results indicated that control overGM-CSF exposure was needed to both recruit and program DCs in vivo. IfGM-CSF alone is to be used for both purposes, its local concentration isdesigned to decrease over time in order to release DCs that becometrapped in the material. Alternatively, provision of a danger signal(e.g., CpG-ODN) in the local environment is used to release DCs fromGM-CSF inhibition once they reside at the infection-mimicking site.

Based on this understanding, a macroporous poly-lactide-co-glycolide(PLG) matrix was designed to present GM-CSF, danger signals, and cancerantigens in a defined spatiotemporal manner in vivo, and serve as aresidence for recruited DCs as they are programmed. GM-CSF wasencapsulated (54% efficiency) into PLG scaffolds using a high pressureCO₂ foaming process. These matrices released approximately 60% of theirbioactive GM-CSF load within the first 5 days, followed by slow andsustained release of bioactive GM-CSF over the next 10 days. Thisrelease profile allows diffusion of the factor through the surroundingtissue to effectively recruit resident DCs.

As described herein, in situ dendritic cell targeting systems areutilized to therapeutically manipulate the immune system with TLRagonists. As described in detail below, macroporous polymeric scaffoldswere designed that deliver three different classes of TLR agonists invivo: CpG-ODN, MPLA, and P(I:C) in combination with GM-CSF, Flt3L, orCCL20 to augment DC recruitment and activation. The ability of in situTLR presentation from macroporous matrices to modulate DC subsetgeneration, and cancer vaccine efficacy in a B16-F10 melanoma model wasalso characterized. The ability of these systems to effect immuneprotection and tumor regression required CD8(+) DCs and correlatedstrongly with plasmacytoid DCs(pDCs) and IL-12 production, regardless ofthe TLR agonist type or dose. Thus, the results presented hereindemonstrate that 3D polymer matrices are utilized to regulate DC subsetsin situ for immunotherapy and indicate that CD8(+) DCs, pDCs and IL-12signaling are critical components of successful material-basedvaccination protocols.

The generation of immunity requires collaboration between dendriticcells (DCs) and T cells, as the priming of cytotoxic T lymphocyte (CTL)by DCs is a crucial event in the fight against infection and tumors(Lanzavecchia A. and Sallusto F., 2001 Cell, 106: 263-266). DCs regulateimmune responses by recognizing, processing, and decoding pathogenassociated molecular patterns (PAMPs) and antigenic molecules(Banchereau J, and Steinman R M., 1998 Nature, 392: 245-252; Mellman I.and Steinman R. M., 2001 Cell, 106: 255-258; Sansonetti P. J., 2006 Nat.Immunol., 7: 1237-1242; Meylan et al., 2006 Nature, 442: 39-44; Akira etal., 2006 Cell, 124: 783-801). PAMP recognition by pattern recognitionreceptors (PRRs) present intercellularly or at the DC surface signal thepresence of infection and triggers signal transduction pathwaysultimately resulting in DC activation (Sansonetti P. J., 2006 Nat.Immunol., 7: 1237-1242; Meylan et al., 2006 Nature, 442: 39-44; Akira etal., 2006 Cell, 124: 783-801). Generally, activated DCs arecharacterized by enhanced expression of MHC and co-stimulatory moleculesand proinflammatory cytokines, which enables DCs to translate pathogenicsignals to naïve T cells and trigger adaptive immune responses(Banchereau J, and Steinman R M., 1998 Nature, 392: 245-252; Mellman I.and Steinman R. M., 2001 Cell, 106: 255-258; Sansonetti P. J., 2006 Nat.Immunol., 7: 1237-1242; Meylan et al., 2006 Nature, 442: 39-44; Akira etal., 2006 Cell, 124: 783-801; Gilboa, E., 2007 J Clin Invest., 117:1195-1203; Banchereau J. and Steinman R. M., 2007 Nature, 49: 419-426).DCs act as a network of distinct subsets that perform specializedfunctions to stimulate and polarize T cell responses in order tocoordinate immune regulation (Naik et al., 2007 Nat Immunol, 8:1217-1226; O'Garral A. and Trinchieri G. 2004 Nat Immunol, 5: 1206-1208;D'Amico A and Wu L., 2003 J Exp Med, 2: 293-303; Villadangos J A andSchnorrer P, 2007 Nat Rev Immunol, 7: 543-555; Liu Y J, 2001 Cell, 106:259-262; Jego et al., 2003 Immunity, 19: 225-234; Randolph et al., 2008Annu. Rev. Immunol., 26: 293-316). Antigen processing and presentationto T cells is predominantly attributed to the conventional DC subset(cDCs), consisting of both CD8(−) DCs and CD8(+) DCs. CD8(+) DCs areespecially adept at cross-presentation of exogenous antigen, IL-12production and induction of cytotoxic T cell responses (Schnorrer P,2006 PNAS 28: 10729-34; Skokos D. and Nussenzweig M. C., J Exp Med, 204:1525-1531; Den Haana et al., 2000 J Exp Med, 12: 1685-1696; Moser M. andMurphy K. M., 2000 Nat. Immunol., 1: 199-205; Hildner et al., 2008Science, 322:1097-1100). The plasmacytoid DC (pDC) subset has thecapacity to produce significant amounts of type-1 interferons (IFNs) inresponse to microbial nucleic acids, particularly during viralinfection, to facilitate T cell activation, growth and survival fordisease clearance (Liu Y J, 2001 Cell, 106: 259-262; Jego et al., 2003Immunity, 19: 225-234; Randolph et al., 2008 Annu. Rev. Immunol., 26:293-316; Kanzler et al., 2007 Nat. Med., 13: 552-559). Moreover, theprocesses mediated by pDC and CD8(+) DC subsets have been associatedwith priming t-helper 1 (Th1) effector cells for the control ofinfection and tumors. A balanced distribution of activated DC subsets isassociated with the control of autoimmune disease and tumors, indicatingthat these cells may cooperate during the generation of protectiveimmunity.

Prior to the invention described herein, cancer vaccines are designed tointroduce antigen in combination with immunostimulatory signals toactivate DCs either ex vivo prior to administration, or in situ (GilboaE., 2007 J Clin Invest., 117: 1195-1203; Banchereau J. and Steinman R.M., 2007 Nature, 49: 419-426; Kanzler et al., 2007 Nat. Med., 13:552-559; Hansen et al., 2013 Vaccine, 31(4), 639-46; Schuler et al.,2003 Curr Opin Immunol, 15: 138-147; Curiel T. J, 2002 J Clin Invest,109: 311-312). A range of stimuli are used to trigger DC maturation anddifferentiation including proinflammatory cytokines, PAMPs recognized bythe toll-like receptor (TLR) family, and feedback signals from innateand adaptive immune cells. As described in detail below, discretecombinations of these stimuli and DC subsets differentially control Tcell activation and polarization, and these components are optimized andexploited to generate effective immune responses that eradicate tumorsor infectious agents. However, it is currently unclear what componentsand DC subsets should be included in cancer vaccines, partly becausecurrent techniques limit the cell types that can be cultured or targeted(Kanzler et al., 2007 Nat. Med., 13: 552-559; Hansen et al., 2013Vaccine, 31(4), 639-46; Schuler et al., 2003 Curr Opin Immunol, 15:138-147; Curiel T. J, 2002 J Clin Invest, 109: 311-312). StandardDC-based protocols widely used in the clinic utilize monocyte-derivedconventional DCs that are unable to cross-present antigens, orefficiently produce IL-12 or type-1 IFNs that can prime CTL-mediatedimmune responses and tumor cell death (Hansen et al., 2013 Vaccine,31(4), 639-46; Schuler et al., 2003 Curr Opin Immunol, 15: 138-147;Curiel T. J, 2002 J Clin Invest, 109: 311-312). Prior to the inventiondescribed herein, there were attempts to utilize type 1 differentiatedDCs in combination with TLR agonists to boost CTL priming capacity, butthis maturation is accompanied by decreased migratory and stimulatoryfunction upon implantation (Hansen et al., 2013 Vaccine, 31(4), 639-46).Described herein are macroporous polymer matrices that regulate thetrafficking and activation of DCs in vivo by precisely controlling thepresentation of GMCSF and CpG-oligonucleotide (CpG-ODN) adjuvants (Aliet al., 2009 Nat Mater, 2: 151-8; Ali et al., 2009 Sci Transl Med,1:8-19). When applied as cancer vaccines, these matrices led to induceCTL-mediated eradication of melanoma tumors (Ali et al., 2009 Sci TranslMed, 1:8-19).

As described herein, matrices were modified to present 3 differentclasses of TLR agonists, CpG-ODN, monophosphoryl lipid A (MPLA), andpolyinosinic:polycytidylic acid (P(I:C)), all in combination withGM-CSF. The ability of each vaccine to recruit and generate activated DCsubsets, in vivo was first quantified. The impact of DC induction on Tcell-mediated immunity and cancer vaccine efficacy in vaccine models ofB16-F10 melanoma was next assessed. These studies demonstrate thatanti-tumor efficacy requires CD8(+) DCs and is strongly correlated withpDC numbers and local IL-12 production. Survival outcomes were alsocorrelated to an array of inflammatory cytokines, which revealed astrong relationship between IL-12 production and antitumor efficacy.Altogether, the results presented herein demonstrate that various DCsubsets are recruited and utilized for in situ vaccination, and provideimportant cellular and molecular insights into cancer vaccine design.

Inflammatory Mediators

Dendritic Cell (DC) proliferation, migration and maturation aresensitive to inflammatory mediators, and granulocyte macrophage colonystimulating factor (GM-CSF) has been identified as a potent stimulatorof immune responses, specifically against cancer antigens. GM-CSF alsohas the ability to recruit and program these antigen-presenting immunecells. Additionally, Cytosine-guanosine (CpG) oligonucleotide (CpG-ODN)sequences found in bacterial DNA are potent immunomodulators thatstimulate DC activation, leading to specific T-cell responses. Creatingan infection mimicking microenvironment by the presentation of exogenousGM-CSF and CpG-ODN provides an avenue to precisely control the numberand timing of DC migration and modulate antigen specific immuneresponses.

The vertebrate immune system employs various mechanisms for pathogenrecognition making it adept at generating antigen-specific responses andclearing infection Immunity is controlled by antigen presenting cells(APCs), especially dendritic cells (DCs), which capture antigens and areactivated by stimuli, unique ‘danger signals’ of the invading pathogen,such as CpG dinucleotide sequences in bacterial DNA (Banchereau J, andSteinman R M. Nature. 392, 245-252. (1998); Klinman D M. Nat. Rev.Immunol. 4, 249-58 (2004); each herein incorporated by reference).

However, cancerous cells, derived from self-tissues, are void of thedanger signals required to signal DC maturation and instead promote animmunosuppressive microenvironment that allows cells to escape immunity.Key elements of infection are inflammatory cytokines and danger signals(FIG. 1). A polymeric material system is ideal to present these factorsin the required spatiotemporal manner to provide an infection-mimickingmicroenvironment in situ that useful as a vaccine. These infectionmimics provide the continuous programming of host DCs, providing forefficient DC activation and dispersement in situ. Theseinfection-mimicking devices are used for numerous vaccine applicationsincluding melanoma cancer vaccines.

In many infections, inflammatory cytokines and danger signals stimulatespecific DC responses that mediate immune recognition and pathogenclearance (FIG. 1). For example, upon bacterial invasion and release oftoxins, skin cells such as fibroblasts, keratinocytes and melanocytesare damaged resulting in the release of inflammatory cytokines, such asGM-CSF (Hamilton J. Trends in Immunol. 23, 403-408. (2002); Hamilton J.,and Anderson G. Growth Factors. 22(4), 225-231. (2004); each hereinincorporated by reference), that act to recruit Langerhans DC (skin) andDC precursors (monocytes; blood) (Hamilton J. Trends in Immunol. 23,403-408. (2002); Hamilton J., and Anderson G. Growth Factors. 22(4),225-231. (2004); Bowne W. B., et al. Cytokines Cell Mol Ther. 5(4),217-25. (1999).; Dranoff, G. Nat. Rev. Cancer 4, 11-22 (2004); eachherein incorporated by reference). As DCs arrive to the site ofinfection they begin to differentiate, and increase in phagocyticability in response to the inflammation (Mellman I., and Steinman R. M.Cell. 106, 255-258. (2001), herein incorporated by reference), and DCsthat ingest bacteria or their products begin to process antigens and DCmaturation proceeds via endosomal TLR9 signaling stimulated by CpGdinucleotide sequences in bacterial DNA (Krieg A. M., Hartmann G., andWeiner G. J. CpG DNA: A potent signal for growth, activation, andmaturation of human dendritic cells. Proc Natl Acad Sci USA. 16,9305-9310 (1999), herein incorporated by reference). Mature DCs thenhome to the lymph nodes where they prime antigen specific T-cellresponses that clear infection.

CpG-ODNs are potent “danger signals” that upregulate DC expression ofCCR7, CD80/86 costimulatory molecules, and MHC-antigen complexesImportantly, TLR9 signaling induces DCs into promoting Th1-like,cytotoxic-T cell responses, by cytokine production (e.g. type 1 IFN) andcross-presentation of antigen onto MHCI molecules. The presentation ofthese signals concurrently with tumor antigens provides the dangersignal needed to promote immune responses that effectively fightcancerous cells.

Different classes of CPG-ODNs promote different immune responsesdepending on the ODN's specific structure and sequence. The ODN utilizedin the present invention, CpG-ODN 1826, has been successfully tested invarious mouse vaccination models, including melanoma. CpG-ODN 1826 hasshown a beneficial effect alone or when used as adjuvant for peptidevaccines and whole cell vaccines. Moreover, ODN 1826 has been shown todirectly promote DC maturation and cytokine production. This particularCpG ODN sequence also indirectly activates Th1 cells and NK cells and,thus, enhances adaptive cellular immune responses.

Vector systems that promote CpG internalization into DCs to enhancedelivery and its localization to TLR9 have been developed. Theamine-rich polycation, polyethylimine (PEI) has been extensively used tocondense plasmid DNA, via association with DNA phosphate groups,resulting in small, positively charge condensates facilitating cellmembrane association and DNA uptake into cells (Godbey W. T., Wu K. K.,and Mikos, A. G. J. of Biomed Mater Res, 1999, 45, 268-275; Godbey W.T., Wu K. K., and Mikos, A. G. Proc Natl Acad Sci USA. 96(9), 5177-81.(1999); each herein incorporated by reference). Consequently, PEI hasbeen utilized as a non-viral vector to enhance gene transfection and tofabricate PEI-DNA loaded PLG matrices that promoted long-term geneexpression in host cells in situ (Huang Y C, Riddle F, Rice K G, andMooney D J. Hum Gene Ther. 5, 609-17. (2005), herein incorporated byreference). Therefore, CpG-ODNs were condensed with PEI molecules, andthe size and charge of these PEI-CpG-ODN condensates, as dependent onthe amine-phosphate charge ratio, was characterized. The ability of PEIcondensation to enhance DC internalization of CpG-ODN was assessed, andthe subsequent decondensation of PEI-CpG-ODN within DCs and itspromotion of DC activation was analyzed in vitro. To determine whetherPEI-CpG-ODNs had the potential to improve upon the vaccination effectsof the GM-CSF based system described in chapter 3, its stimulatoryeffects on DCs maturation and mobilization in the presence of GM-CSF wasalso examined.

To appropriately mimic infection and program cells in situ a PLG systemwas designed to not only serve as a drug delivery device, that releasesinflammatory cytokines (e.g. GM-CSF) but also as a physical structure towhich the DCs are recruited and reside while they are activated bydanger signals (e.g. CpG-ODNs). The ability to control DC recruitment toand DC residence within porous PLG matrices is achieved using temporalcontrol over the delivery of GM-CSF in situ, which results in batches ofprogrammed DCs being dispersed only when GM-CSF levels were designed tosubside in situ. This system dispersed 6% of programmed DCs to the lymphnodes and induced protective anti-tumor immunity in 23% of mice whenapplied as a cancer vaccine. The cell programming and dispersementefficiency is improved using an overriding secondary signal (CpG-ODN)that continuously releases DCs from GM-CSF inhibition and promotes DCmaturation and dispersement in the presence of high GM-CSF levels insitu. Specifically, PLG matrices were fabricated to locally presentsynthetic CpG-ODN with exogenous GM-CSF allowing for DCs recruited byGM-CSF to be stimulated by CpG-ODN in situ.

Dendritic Cells

Dendritic cells (DCs) are immune cells within the mammalian immunesystem and are derived from hematopoietic bone marrow progenitor cells.More specifically, dendritic cells can be categorized into lymphoid (orplasmacytoid) dendritic cell (pDC) and myeloid dendritic cell (mDC)subdivisions having arisen from a lymphoid (or plasmacytoid) or myeloidprecursor cell, respectively. From the progenitor cell, regardless ofthe progenitor cell type, an immature dendritic cell is born. Immaturedendritic cells are characterized by high endocytic activity and lowT-cell activation potential. Thus, immature dendritic cellsconstitutively sample their immediate surrounding environment forpathogens. Exemplary pathogens include, but are not limited to, a virusor a bacteria. Sampling is accomplished by pattern recognition receptors(PRRs) such as the toll-like receptors (TLRs). Dendritic cells activateand mature once a pathogen is recognized by a pattern recognitionreceptor, such as a toll-like receptor.

Mature dendritic cells not only phagocytose pathogens and break themdown, but also, degrade their proteins, and present pieces of theseproteins, also referred to as antigens, on their cell surfaces using MHC(Major Histocompatibility Complex) molecules (Classes I, II, and III).Mature dendritic cells also upregulate cell-surface receptors that serveas co-receptors for T-cell activation. Exemplary co-receptors include,but are not limited to, CD80, CD86, and CD40. Simultaneously, maturedendritic cells upregulate chemotactic receptors, such as CCR7, thatallows the cell to migrate through the blood stream or the lymphaticsystem to the spleen or lymph node, respectively.

Dendritic cells are present in external tissues that are in contact withthe external environment such as the skin (dendritic cells residing inskin are also referred to as Langerhans cells). Alternatively, dendriticcells are present in internal tissues that are in contact with theexternal environment such as linings of the nose, lungs, stomach, andintestines. Finally, immature dendritic cells reside in the bloodstream. Once activated, dendritic cells from all off these tissuesmigrate to lymphoid tissues where they present antigens and interactwith T cells and B cells to initiate an immune response. One signalingsystem of particular importance for the present invention involves thechemokine receptor CCR7 expressed on the surface of dendritic cells andthe chemokine receptor ligand CCL19 secreted by lymph node structures toattract migrating mature dendritic cells toward high concentrations ofimmune cells. Exemplary immune cells activated by contact with maturedendritic cells include, but are not limited to, helper T cells, killerT cells, and B cells. Although multiple cell types within the immunesystem present antigens, including macrophages and B lymphocytes,dendritic cells are the most potent activators of all antigen-presentingcells.

Dendritic cells earned their name from the characteristic cell shapecomprising multiple dendrites extending from the cell body. Thefunctional benefit of this cell shape is a significantly increased cellsurface and contact area to the surroundings compared to the cellvolume. Immature dendritic cells sometimes lack the characteristicdendrite formations and are referred to as veiled cells. Veiled cellspossess large cytoplasmic veils rather than dendrites.

Plasmacytoid dendritic cells (pDCs) are innate immune cells thatcirculate in the blood and are found in peripheral lymphoid organs. Theyconstitute <0.4% of peripheral blood mononuclear cells (PBMC). In humansthese cells express the surface markers CD123, BDCA-2(CD303) andBDCA-4(CD304), but do not express high levels of CD11c or CD14, whichdistinguishes them from conventional dendritic cells or monocytes,respectively. Mouse pDC express CD11c, B220, BST-2 (mPDCA) and Siglec-Hand are negative for CD11b. As components of the innate immune system,these cells express intracellular Toll-like receptors 7 and 9 whichdetect ssRNA and CpG DNA motifs, respectively. Upon stimulation andsubsequent activation, these cells produce large amounts of type Iinterferon (mainly IFN-α (alpha) and IFN-β (beta)), which are criticalpleiotropic anti-viral compounds mediating a wide range of effects. TheCD8− subset presents antigen using the class II pathway to CD4+ helper Tcells. The CD8+ subset presents antigens using the class I pathway. Thepeptide/MHC class I molecules are presented to CD8+ T cells which go onto become cytotoxic T lymphocytes (CTL). The CD8 cell surface protein inthe mouse corresponds to the CD141 cell surface protein in the human.CD8/CD141-positive cells express TLR3 and are preferentially activatedby TLR3 agonists.

Toll-Like Receptors (TLRs)

TLRs are a class of single transmembrane domain, non-catalytic,receptors that recognize structurally conserved molecules referred to aspathogen-associated molecular patterns (PAMPs). PAMPs are present onmicrobes and are distinguishable from host molecules. TLRs are presentin all vertebrates. Thirteen TLRs (referred to as TLRs1-13,consecutively) have been identified in humans and mice. Humans compriseTLRs 1-10.

TLRs and interleukin-1 (IL-1) receptors comprise a receptor superfamilythe members of which all share a TIR domain (Toll-IL-1 receptor). TIRdomains exist in three varieties with three distinct functions. TIRdomains of subgroup 1 are present in receptors for interleukins producedby macrophages, monocytes, and dendritic cells. TIR domains of subgroup2 are present in classical TLRs which bind directly or indirectly tomolecules of microbial origin. TIR domains of subgroup 3 are present incytosolic adaptor proteins that mediate signaling between proteinscomprising TIR domains of subgroups 1 and 2.

TLR ligands comprise molecules that are constantly associated with andhighly specific for a threat to the host's survival such as a pathogenor cellular stress. TLR ligands are highly specific for pathogens andnot the host. Exemplary pathogenic molecules include, but are notlimited to, lipopolysaccharides (LPS), lipoproteins, lipoarabinomannan,flagellin, double-stranded RNA, and unmethylated CpG islands of DNA.

In one preferred embodiment of the present invention, the Toll-Likereceptor 9 (TLR9) is activated by specific unmethylated CpG-containingsequences in bacterial DNA or synthetic oligonucleotides (ODNs) found inthe endosomal compartment of dendritic cells. Methylation status of theCpG site is a crucial distinction between bacterial and mammalian DNA,as well as between normal and cancerous tissue. Unmethylated ODNsincluding one or more CpG motifs mimic the effects of bacterial DNA.Alternatively, or in addition, unmethylated ODNs including one or moreCpG motifs occur within oncogenes present within malignant tumor cells.

One or more sequences of the TLR-9 receptor recognizes one or moreCpG-ODN sequences of the present invention. TLR-9 receptors encompassedby the present invention are described by the following sequences.

Human TLR-9, isoform A, is encoded by the following mRNA sequence (NCBIAccession No. NM_017442 and SEQ ID NO: 1; the start codon for all mRNAsequences presented herein is bolded and capitalized):

   1 ggaggtcttg tttccggaag atgttgcaag gctgtggtga aggcaggtgc agcctagcct  61 cctgctcaag ctacaccctg gccctccacg catgaggccc tgcagaactc tggagatggt 121 gcctacaagg gcagaaaagg acaagtcggc agccgctgtc ctgagggcac cagctgtggt 181 gcaggagcca agacctgagg gtggaagtgt cctcttagaa tggggagtgc ccagcaaggt 241 gtacccgcta ctggtgctat ccagaattcc catctctccc tgctctctgc ctgagctctg 301 ggccttagct cctccctggg cttggtagag gacaggtgtg aggccctcat gggatgtagg 361 ctgtctgaga ggggagtgga aagaggaagg ggtgaaggag ctgtctgcca tttgactatg 421 caaatggcct ttgactcatg ggaccctgtc ctcctcactg ggggcagggt ggagtggagg 481 gggagctact aggctggtat aaaaatctta cttcctctat tctctgagcc gctgctgccc 541 ctgtgggaag ggacctcgag tgtgaagcat ccttccctgt agctgctgtc cagtctgccc 601 gccagaccct ctggagaagc ccctgccccc cagcATGggt ttctgccgca gcgccctgca 661 cccgctgtct ctcctggtgc aggccatcat gctggccatg accctggccc tgggtacctt 721 gcctgccttc ctaccctgtg agctccagcc ccacggcctg gtgaactgca actggctgtt 781 cctgaagtct gtgccccact tctccatggc agcaccccgt ggcaatgtca ccagcctttc 841 cttgtcctcc aaccgcatcc accacctcca tgattctgac tttgcccacc tgcccagcct 901 gcggcatctc aacctcaagt ggaactgccc gccggttggc ctcagcccca tgcacttccc 961 ctgccacatg accatcgagc ccagcacctt cttggctgtg cccaccctgg aagagctaaa1021 cctgagctac aacaacatca tgactgtgcc tgcgctgccc aaatccctca tatccctgtc1081 cctcagccat accaacatcc tgatgctaga ctctgccagc ctcgccggcc tgcatgccct1141 gcgcttccta ttcatggacg gcaactgtta ttacaagaac ccctgcaggc aggcactgga1201 ggtggccccg ggtgccctcc ttggcctggg caacctcacc cacctgtcac tcaagtacaa1261 caacctcact gtggtgcccc gcaacctgcc ttccagcctg gagtatctgc tgttgtccta1321 caaccgcatc gtcaaactgg cgcctgagga cctggccaat ctgaccgccc tgcgtgtgct1381 cgatgtgggc ggaaattgcc gccgctgcga ccacgctccc aacccctgca tggagtgccc1441 tcgtcacttc ccccagctac atcccgatac cttcagccac ctgagccgtc ttgaaggcct1501 ggtgttgaag gacagttctc tctcctggct gaatgccagt tggttccgtg ggctgggaaa1561 cctccgagtg ctggacctga gtgagaactt cctctacaaa tgcatcacta aaaccaaggc1621 cttccagggc ctaacacagc tgcgcaagct taacctgtcc ttcaattacc aaaagagggt1681 gtcctttgcc cacctgtctc tggccccttc cttcgggagc ctggtcgccc tgaaggagct1741 ggacatgcac ggcatcttct tccgctcact cgatgagacc acgctccggc cactggcccg1801 cctgcccatg ctccagactc tgcgtctgca gatgaacttc atcaaccagg cccagctcgg1861 catcttcagg gccttccctg gcctgcgcta cgtggacctg tcggacaacc gcatcagcgg1921 agcttcggag ctgacagcca ccatggggga ggcagatgga ggggagaagg tctggctgca1981 gcctggggac cttgctccgg ccccagtgga cactcccagc tctgaagact tcaggcccaa2041 ctgcagcacc ctcaacttca ccttggatct gtcacggaac aacctggtga ccgtgcagcc2101 ggagatgttt gcccagctct cgcacctgca gtgcctgcgc ctgagccaca actgcatctc2161 gcaggcagtc aatggctccc agttcctgcc gctgaccggt ctgcaggtgc tagacctgtc2221 ccacaataag ctggacctct accacgagca ctcattcacg gagctaccac gactggaggc2281 cctggacctc agctacaaca gccagccctt tggcatgcag ggcgtgggcc acaacttcag2341 cttcgtggct cacctgcgca ccctgcgcca cctcagcctg gcccacaaca acatccacag2401 ccaagtgtcc cagcagctct gcagtacgtc gctgcgggcc ctggacttca gcggcaatgc2461 actgggccat atgtgggccg agggagacct ctatctgcac ttcttccaag gcctgagcgg2521 tttgatctgg ctggacttgt cccagaaccg cctgcacacc ctcctgcccc aaaccctgcg2581 caacctcccc aagagcctac aggtgctgcg tctccgtgac aattacctgg ccttctttaa2641 gtggtggagc ctccacttcc tgcccaaact ggaagtcctc gacctggcag gaaaccagct2701 gaaggccctg accaatggca gcctgcctgc tggcacccgg ctccggaggc tggatgtcag2761 ctgcaacagc atcagcttcg tggcccccgg cttcttttcc aaggccaagg agctgcgaga2821 gctcaacctt agcgccaacg ccctcaagac agtggaccac tcctggtttg ggcccctggc2881 gagtgccctg caaatactag atgtaagcgc caaccctctg cactgcgcct gtggggcggc2941 ctttatggac ttcctgctgg aggtgcaggc tgccgtgccc ggtctgccca gccgggtgaa3001 gtgtggcagt ccgggccagc tccagggcct cagcatcttt gcacaggacc tgcgcctctg3061 cctggatgag gccctctcct gggactgttt cgccctctcg ctgctggctg tggctctggg3121 cctgggtgtg cccatgctgc atcacctctg tggctgggac ctctggtact gcttccacct3181 gtgcctggcc tggcttccct ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc3241 ctacgatgcc ttcgtggtct tcgacaaaac gcagagcgca gtggcagact gggtgtacaa3301 cgagcttcgg gggcagctgg aggagtgccg tgggcgctgg gcactccgcc tgtgcctgga3361 ggaacgcgac tggctgcctg gcaaaaccct ctttgagaac ctgtgggcct cggtctatgg3421 cagccgcaag acgctgtttg tgctggccca cacggaccgg gtcagtggtc tcttgcgcgc3481 cagcttcctg ctggcccagc agcgcctgct ggaggaccgc aaggacgtcg tggtgctggt3541 gatcctgagc cctgacggcc gccgctcccg ctatgtgcgg ctgcgccagc gcctctgccg3601 ccagagtgtc ctcctctggc cccaccagcc cagtggtcag cgcagcttct gggcccagct3661 gggcatggcc ctgaccaggg acaaccacca cttctataac cggaacttct gccagggacc3721 cacggccgaa tagccgtgag ccggaatcct gcacggtgcc acctccacac tcacctcacc3781 tctgcctgcc tggtctgacc ctcccctgct cgcctccctc accccacacc tgacacagag3841 caggcactca ataaatgcta ccgaaggc

Human TLR-9, isoform A, is encoded by the following amino acid sequence(NCBI Accession No. NP_059138 and SEQ ID NO: 2):

MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNVVLFLKSVPHFSMAAPRGNVTSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFLAVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYYKNPCRQALEVAPGALLGLGNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYNRIVKLAPEDLANLTALRVLDVGGNCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLNASWFRGLGNLRVLDLSENFLYKCITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSFGSLVALKELDMHGIFFRSLDETTLRPLARLPMLQTLRLQMNFINQAQLGIFRAFPGLRYVDLSDNRISGASELTATMGEADGGEKVWLQPGDLAPAPVDTPSSEDFRPNCSTLNFTLDLSRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLDLSHNKLDLYHEHSFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQQLCSTSLRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTLRNLPKSLQVLRLRDNYLAFFKWWSLHFLPKLEVLDLAGNQLKALTNGSLPAGTRLRRLDVSCNSISFVAPGFFSKAKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQAAVPGLPSRVKCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLCGWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE 

Human TLR3 is encoded by the following mRNA sequence (GenBank AccessionNo. NM_003265.2 (GI:19718735), incorporated herein by reference; SEQ IDNO: 5):

   1 cactttcgag agtgccgtct atttgccaca cacttccctg atgaaatgtc tggatttgga  61 ctaaagaaaa aaggaaaggc tagcagtcat ccaacagaat cATGagacag actttgcctt 121 gtatctactt ttgggggggc cttttgccct ttgggatgct gtgtgcatcc tccaccacca 181 agtgcactgt tagccatgaa gttgctgact gcagccacct gaagttgact caggtacccg 241 atgatctacc cacaaacata acagtgttga accttaccca taatcaactc agaagattac 301 cagccgccaa cttcacaagg tatagccagc taactagctt ggatgtagga tttaacacca 361 tctcaaaact ggagccagaa ttgtgccaga aacttcccat gttaaaagtt ttgaacctcc 421 agcacaatga gctatctcaa ctttctgata aaacctttgc cttctgcacg aatttgactg 481 aactccatct catgtccaac tcaatccaga aaattaaaaa taatcccttt gtcaagcaga 541 agaatttaat cacattagat ctgtctcata atggcttgtc atctacaaaa ttaggaactc 601 aggttcagct ggaaaatctc caagagcttc tattatcaaa caataaaatt caagcgctaa 661 aaagtgaaga actggatatc tttgccaatt catctttaaa aaaattagag ttgtcatcga 721 atcaaattaa agagttttct ccagggtgtt ttcacgcaat tggaagatta tttggcctct 781 ttctgaacaa tgtccagctg ggtcccagcc ttacagagaa gctatgtttg gaattagcaa 841 acacaagcat tcggaatctg tctctgagta acagccagct gtccaccacc agcaatacaa 901 ctttcttggg actaaagtgg acaaatctca ctatgctcga tctttcctac aacaacttaa 961 atgtggttgg taacgattcc tttgcttggc ttccacaact agaatatttc ttcctagagt1021 ataataatat acagcatttg ttttctcact ctttgcacgg gcttttcaat gtgaggtacc1081 tgaatttgaa acggtctttt actaaacaaa gtatttccct tgcctcactc cccaagattg1141 atgatttttc ttttcagtgg ctaaaatgtt tggagcacct taacatggaa gataatgata1201 ttccaggcat aaaaagcaat atgttcacag gattgataaa cctgaaatac ttaagtctat1261 ccaactcctt tacaagtttg cgaactttga caaatgaaac atttgtatca cttgctcatt1321 ctcccttaca catactcaac ctaaccaaga ataaaatctc aaaaatagag agtgatgctt1381 tctcttggtt gggccaccta gaagtacttg acctgggcct taatgaaatt gggcaagaac1441 tcacaggcca ggaatggaga ggtctagaaa atattttcga aatctatctt tcctacaaca1501 agtacctgca gctgactagg aactcctttg ccttggtccc aagccttcaa cgactgatgc1561 tccgaagggt ggcccttaaa aatgtggata gctctccttc accattccag cctcttcgta1621 acttgaccat tctggatcta agcaacaaca acatagccaa cataaatgat gacatgttgg1681 agggtcttga gaaactagaa attctcgatt tgcagcataa caacttagca cggctctgga1741 aacacgcaaa ccctggtggt cccatttatt tcctaaaggg tctgtctcac ctccacatcc1801 ttaacttgga gtccaacggc tttgacgaga tcccagttga ggtcttcaag gatttatttg1861 aactaaagat catcgattta ggattgaata atttaaacac acttccagca tctgtcttta1921 ataatcaggt gtctctaaag tcattgaacc ttcagaagaa tctcataaca tccgttgaga1981 agaaggtttt cgggccagct ttcaggaacc tgactgagtt agatatgcgc tttaatccct2041 ttgattgcac gtgtgaaagt attgcctggt ttgttaattg gattaacgag acccatacca2101 acatccctga gctgtcaagc cactaccttt gcaacactcc acctcactat catgggttcc2161 cagtgagact ttttgataca tcatcttgca aagacagtgc cccctttgaa ctctttttca2221 tgatcaatac cagtatcctg ttgattttta tctttattgt acttctcatc cactttgagg2281 gctggaggat atctttttat tggaatgttt cagtacatcg agttcttggt ttcaaagaaa2341 tagacagaca gacagaacag tttgaatatg cagcatatat aattcatgcc tataaagata2401 aggattgggt ctgggaacat ttctcttcaa tggaaaagga agaccaatct ctcaaatttt2461 gtctggaaga aagggacttt gaggcgggtg tttttgaact agaagcaatt gttaacagca2521 tcaaaagaag cagaaaaatt atttttgtta taacacacca tctattaaaa gacccattat2581 gcaaaagatt caaggtacat catgcagttc aacaagctat tgaacaaaat ctggattcca2641 ttatattggt tttccttgag gagattccag attataaact gaaccatgca ctctgtttgc2701 gaagaggaat gtttaaatct cactgcatct tgaactggcc agttcagaaa gaacggatag2761 gtgcctttcg tcataaattg caagtagcac ttggatccaa aaactctgta cattaaattt2821 atttaaatat tcaattagca aaggagaaac tttctcaatt taaaaagttc tatggcaaat2881 ttaagttttc cataaaggtg ttataatttg tttattcata tttgtaaatg attatattct2941 atcacaatta catctcttct aggaaaatgt gtctccttat ttcaggccta tttttgacaa3001 ttgacttaat tttacccaaa ataaaacata taagcacgta aaaaaaaaaa aaaaaaa

Human TLR3 is encoded by the following amino acid sequence (GenBankAccession No. ABC86910.1 (GI:86161330), incorporated herein byreference; SEQ ID NO: 4):

  1 mrqtlpciyf wggllpfgml cassttkctv shevadcshl kltqvpddlp tnitvlnlth 61 nqlrrlpaan ftrysqltsl dvgfntiskl epelcqklpm lkvlnlqhne lsqlsdktfa121 fctnltelhl msnsiqkikn npfvkqknli tldlshngls stklgtqvql enlqelllsn181 nkiqalksee ldifansslk klelssnqik efspgcfhai grlfglflnn vqlgpsltek241 lclelantsi rnlslsnsql sttsnttflg lkwtnltmld lsynnlnvvg ndsfawlpql301 eyffleynni qhlfshslhg lfnvrylnlk rsftkqsisl aslpkiddfs fqwlkclehl361 nmedndipgi ksnmftglin lkylslsnsf tslrtltnet fvslahsplh ilnltknkis421 kiesdafswl ghlevldlgl neiggeltgq ewrglenife iylsynkylq ltrnsfalvp481 slqrlmlrrv alknvdssps pfqplrnlti ldlsnnnian inddmlegle kleildlqhn541 nlarlwkhan pggpiyflkg lshlhilnle sngfdeipve vfkdlfelki idlglnnlnt601 lpasvfnnqv slkslnlqkn litsvekkvf gpafrnltel dmrfnpfdct cesiawfvnw661 inethtnipe lsshylcntp phyhgfpvrl fdtssckdsa pfelffmint sillififiv721 llihfegwri sfywnvsvhr vlgfkeidrq teqfeyaayi ihaykdkdwv wehfssmeke781 dqslkfclee rdfeagvfel eaivnsikrs rkiifvithh llkdpickrf kvhhavqqai841 eqnldsiilv fleeipdykl nhalclrrgm fkshcilnwp vqkerigafr hklqvalgsk901 nsvh

The nucleic acid sequence of human TLR1 is provided in GenBank AccessionNo. NM_003263.3 (GI:41350336), incorporated herein by reference. Theamino acid sequence of human TLR1 is provided in GenBank Accession No.NP_003254.2 (GI:41350337), incorporated herein by reference.

The nucleic acid sequence of human TLR2 is provided in GenBank AccessionNo. NM_003264.3 (GI:68160956), incorporated herein by reference. Theamino acid sequence of human TLR2 is provided in GenBank Accession No.NP_003255.2 (GI:19718734), incorporated herein by reference.

The nucleic acid sequence of human TLR4 is provided in GenBank AccessionNo. NM_138554.4 (GI:373432600), incorporated herein by reference. Theamino acid sequence of human TLR4 is provided in GenBank Accession No.NP_612564.1 (GI:19924149), incorporated herein by reference.

The nucleic acid sequence of human TLR5 is provided in GenBank AccessionNo. NM_003268.5 (GI:281427130), incorporated herein by reference. Theamino acid sequence of human TLR5 is provided in GenBank Accession No.NP_003259.2 (GI:16751843), incorporated herein by reference.

The nucleic acid sequence of human TLR6 is provided in GenBank AccessionNo. NM_006068.4 (GI:318067953), incorporated herein by reference. Theamino acid sequence of human TLR6 is provided in GenBank Accession No.NP_006059.2 (GI:20143971), incorporated herein by reference.

The nucleic acid sequence of human TLR7 is provided in GenBank AccessionNo. NM_016562.3 (GI:67944638), incorporated herein by reference. Theamino acid sequence of human TLR7 is provided in GenBank Accession No.NP_057646.1 (GI:7706093), incorporated herein by reference.

The nucleic acid sequence of human TLR8 is provided in GenBank AccessionNo. NM_138636.4 (GI:257196253), incorporated herein by reference. Theamino acid sequence of human TLR8 is provided in GenBank Accession No.NP_619542.1 (GI:20302168), incorporated herein by reference.

The nucleic acid sequence of human TLR10 is provided in GenBankAccession No. NM_030956.3 (GI:306140488), incorporated herein byreference. The amino acid sequence of human TLR10 is provided in GenBankAccession No. NP_112218.2 (GI:62865618), incorporated herein byreference.

The nucleic acid sequence of mouse TLR11 is provided in GenBankAccession No. NM_205819.3 (GI:408684412), incorporated herein byreference. The amino acid sequence of mouse TLR11 is provided in GenBankAccession No. NP_991388.2 (GI:408684413), incorporated herein byreference.

The nucleic acid sequence of mouse TLR12 is provided in GenBankAccession No. NM_205823.2 (GI:148539900), incorporated herein byreference. The amino acid sequence of mouse TLR12 is provided in GenBankAccession No. NP_991392.1 (GI:45430001), incorporated herein byreference.

The nucleic acid sequence of mouse TLR13 is provided in GenBankAccession No. NM_205820.1 (GI:45429998), incorporated herein byreference. The amino acid sequence of mouse TLR13 is provided in GenBankAccession No. NP_991389.1 (GI:45429999), incorporated herein byreference.

A representative list of TLR agonists (both synthetic and naturalligands), along with their corresponding receptor is provided in Table 2below.

TABLE 2 Receptor Pathogen Associated Ligands (PAMPS) [1] Ligand Naturalhost Synthetic Ligands TLR 1 multiple triacyl lipopeptides BacteriaPam3Cys-* TLR 2 multiple glycolipids Bacteria CFA multiple lipopeptidesBacteria MALP2-** multiple lipoproteins Bacteria Pam2Cys** lipoteichoicacid Gram Positive Bacteria FSL-1 HSP 70, or other heat shock proteinsHost cells Hib-OMPC zymosan (Beta- glucan) Fungi Numerous others TLR 3Double stranded RNA viruses Poly (I:C); Low and High molecular weightPoly (A:U) TLR 4 lipopolysacharides (LPS); or LPS derivatives such Gramnegative bacteria AGP as MPLA several heat shock proteins Bacteria andhost cells MPLA fibrinogen host cells RC-529 heparin sulfate fragmentshost cells MDF2β hyaluronic acid fragments host cells CFA nickel Variousopoid drugs TLR 5 Flagellin Bacteria Flagellin TLR 6 multiple diacyllipopeptides Mycoplasma FSL1-** Pam2Cys** MALP2-** TLR 7 Viral ssRNA(Influenza, VSV, HIV, HCV) RNA viruses Guanosine analogs;imidazoquinolines (e.g. Imiquimod, Aldara ® R848, Resiquimod ®),Loxorbine TLR 8 small synthetic compounds; single-stranded RNA RNA,Human and viral Imidazoquinoline; Loxoribine; ssPolyU, 3M-012 TLR 9Unmethylated CpG Oligodeoxynucleotide DNA Bacteria, DNA virusesCpG-oligonucleotides, numerous DNA; dsDNA viruses (HSV, MCMV); Hemozoinsequences have been synthesized (Plasmodium) (e.g CpG-ODN 2006, 1826,2395) TLR 10 unknown TLR 11 Profilin Toxoplasma gondii TLR 12 ProfilinToxoplasma gondii TLR 13 [2][3] bacterial ribosomal RNA sequence Virus,bacteria “CGGAAAGACC” (SEQ ID NO: 13) *Ligands recognized by TLR1 andTLR2 **Ligands recognized by TLR2 and TLR6 References Meyer T,Stockfleth E. Clinical investigational of Toll-like receptor agonists.Expert opinion on investigational drugs. 2008; 17: 1051-1065. [PubMed]van Duin D, Medzhitov R, Shaw A C. Triggering TLR signaling invaccination. Trends in immunology. 2006; 27: 49-55 Kumar H, Kawai T,Akira Toll-like receptors and innate immunity. Biochemical andbiophysical research communications. 2009; 388: 621-625. Waltenbaugh C,Doan T, Melvoid R, Viselli S (2008). Immunology. Lippincott'sIllustrated reviews. Philadelphia: Wolters Kluwer Health/LippincottWilliams & Wilkins. pp. 17. Shi Z, Cai Z, Sanchez A, et al. (February2011). A novel Toll-like receptor that recognizes vesicular stomatitisvirus. 286. pp. 4517-24. Oldenburg M. Kruger A, A, Ferstl R, et al.(August 2012). TLR1S recognizes bacterial 23S rRNA devoid of erthromycinresistance-forming modification. 337. pp. 111-5, S. Gnjatic, N. B.Sawhney, N. Bhardwaj Toll-like receptor agonists: are they goodadjuvants? Cancer J., 16 (4) (2010), pp. 382-391.

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a proteinsecreted by macrophages, T cells, mast cells, endothelial cells andfibroblasts. Specifically, GM-CSF is a cytokine that functions as awhite blood cell growth factor. GM-CSF stimulates stem cells to producegranulocytes and monocytes. Monocytes exit the blood stream, migrateinto tissue, and subsequently mature into macrophages.

Scaffold devices described herein comprise and release GM-CSFpolypeptides to attract host DCs to the device. Contemplated GM-CSFpolypeptides are isolated from endogenous sources or synthesized in vivoor in vitro. Endogenous GM-CSF polypeptides are isolated from healthyhuman tissue. Synthetic GM-CSF polypeptides are synthesized in vivofollowing transfection or transformation of template DNA into a hostorganism or cell, e.g. a mammal or cultured human cell line.Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro bypolymerase chain reaction (PCR) or other art-recognized methodsSambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3(1989), herein incorporated by reference). [01] GM-CSF polypeptides aremodified to increase protein stability in vivo. Alternatively, GM-CSFpolypeptides are engineered to be more or less immunogenic. Endogenousmature human GM-CSF polypeptides are glycosylated, reportedly, at aminoacid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (seeU.S. Pat. No. 5,073,627). GM-CSF polypeptides of the present inventionare modified at one or more of these amino acid residues with respect toglycosylation state.

GM-CSF polypeptides are recombinant. Alternatively GM-CSF polypeptidesare humanized derivatives of mammalian GM-CSF polypeptides. Exemplarymammalian species from which GM-CSF polypeptides are derived include,but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat,dog, monkey, or primate. In a preferred embodiment, GM-CSF is arecombinant human protein (PeproTech, Catalog #300-03). Alternatively,GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog#315-03). Finally, GM-CSF is a humanized derivative of a recombinantmouse protein.

Human Recombinant GM-CSF (PeproTech, Catalog #300-03) is encoded by thefollowing polypeptide sequence (SEQ ID NO: 3):

MAPARSPSPS TQPWEHVNAI QEARRLLNLS RDTAAEMNETVEVISEMFDL QEPTCLQTRL ELYKQGLRGS LTKLKGPLTMMASHYKQHCP PTPETSCATQ IITFESFKEN LKDFLLVIPF DCWEPVQE

Murine Recombinant GM-CSF (PeproTech, Catalog #315-03) is encoded by thefollowing polypeptide sequence (SEQ ID NO: 7):

MAPTRSPITV TRPWKHVEAI KEALNLLDDM PVTLNEEVEVVSNEFSFKKL TCVQTRLKIF EQGLRGNFTK LKGALNMTASYYQTYCPPTP ETDCETQVTT YADFIDSLKT FLTDIPFECK KPVQK

Human Endogenous GM-CSF is encoded by the following mRNA sequence (NCBI

Accession No. NM_000758 and SEQ ID NO: 8):

  1 acacagagag aaaggctaaa gttctctgga ggatgtggct gcagagcctg ctgctcttgg 61 gcactgtggc ctgcagcatc tctgcacccg cccgctcgcc cagccccagc acgcagccct121 gggagcatgt gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg181 ctgctgagat gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga241 cctgcctaca gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc301 tcaagggccc cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg361 aaacttcctg tgcaacccag attatcacct ttgaaagttt caaagagaac ctgaaggact421 ttctgcttgt catccccttt gactgctggg agccagtcca ggagtgagac cggccagatg481 aggctggcca agccggggag ctgctctctc atgaaacaag agctagaaac tcaggatggt541 catcttggag ggaccaaggg gtgggccaca gccatggtgg gagtggcctg gacctgccct601 gggccacact gaccctgata caggcatggc agaagaatgg gaatatttta tactgacaga661 aatcagtaat atttatatat ttatattttt aaaatattta tttatttatt tatttaagtt721 catattccat atttattcaa gatgttttac cgtaataatt attattaaaa atatgcttct781 a

Human Endogenous GM-CSF is encoded by the following amino acid sequence

(NCBI Accession No. NP_000749.2 and SEQ ID NO: 9):

MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

Cytosine-Guanosine (CpG) Oligonucleotide (CpG-ODN) Sequences

CpG sites are regions of deoxyribonucleic acid (DNA) where a cysteinenucleotide occurs next to a guanine nucleotide in the linear sequence ofbases along its length (the “p” represents the phosphate linkage betweenthem and distinguishes them from a cytosine-guanine complementary basepairing). CpG sites play a pivotal role in DNA methylation, which is oneof several endogenous mechanisms cells use to silence gene expression.Methylation of CpG sites within promoter elements can lead to genesilencing. In the case of cancer, it is known that tumor suppressorgenes are often silences while oncogenes, or cancer-inducing genes, areexpressed Importantly, CpG sites in the promoter regions of tumorsuppressor genes (which prevent cancer formation) have been shown to bemethylated while CpG sites in the promoter regions of oncogenes arehypomethylated or unmethylated in certain cancers. The TLR-9 receptorbinds unmethylated CpG sites in DNA.

The present invention comprises CpG dinucleotides and oligonucleotides.Contemplated CpG oligonucleotides are isolated from endogenous sourcesor synthesized in vivo or in vitro. Exemplary sources of endogenous CpGoligonucleotides include, but are not limited to, microorganisms,bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively,endogenous CpG oligonucleotides are isolated from mammalian benign ormalignant neoplastic tumors. Synthetic CpG oligonucleotides aresynthesized in vivo following transfection or transformation of templateDNA into a host organism. Alternatively, Synthetic CpG oligonucleotidesare synthesized in vitro by polymerase chain reaction (PCR) or otherart-recognized methods (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

CpG oligonucleotides are presented for cellular uptake by dendriticcells. In one embodiment, naked CpG oligonucleotides are used. The term“naked” is used to describe an isolated endogenous or syntheticpolynucleotide (or oligonucleotide) that is free of additionalsubstituents. In another embodiment, CpG oligonucleotides are bound toone or more compounds to increase the efficiency of cellular uptake.Alternatively, or in addition, CpG oligonucleotides are bound to one ormore compounds to increase the stability of the oligonucleotide withinthe scaffold and/or dendritic cell.

CpG oligonucleotides are condensed prior to cellular uptake. In onepreferred embodiment, CpG oligonucleotides are condensed usingpolyethylimine (PEI), a cationic polymer that increases the efficiencyof cellular uptake into dendritic cells.

CpG oligonucleotides of the present invention can be divided intomultiple classes. For example, exemplary CpG-ODNs encompassed bycompositions, methods and devices of the present invention arestimulatory, neutral, or suppressive. The term “stimulatory” used hereinis meant to describe a class of CpG-ODN sequences that activate TLR9.The term “neutral” used herein is meant to describe a class of CpG-ODNsequences that do not activate TLR9. The term “suppressive” used hereinis meant to describe a class of CpG-ODN sequences that inhibit TLR9. Theterm “activate TLR9” describes a process by which TLR9 initiatesintracellular signaling.

Stimulatory CpG-ODNs can further be divided into three types A, B and C,which differ in their immune-stimulatory activities. Type A stimulatoryCpG ODNs are characterized by a phosphodiester central CpG-containingpalindromic motif and a phosphorothioate 3′ poly-G string. Followingactivation of TLR9, these CpG ODNs induce high IFN-α production fromplasmacytoid dendritic cells (pDC). Type A CpG ODNs weakly stimulateTLR9-dependent NF-κB signaling.

Type B stimulatory CpG ODNs contain a full phosphorothioate backbonewith one or more CpG dinucleotides. Following TLR9 activation, theseCpG-ODNs strongly activate B cells. In contrast to Type A Cpg-ODNs, TypeB CpG-ODNS weakly stimulate IFN-α secretion.

Type C stimulatory CpG ODNs comprise features of Types A and B. Type CCpG-ODNs contain a complete phosphorothioate backbone and a CpGcontaining palindromic motif. Similar to Type A CpG ODNs, Type C CpGODNs induce strong IFN-α production from pDC. Similar to Type B CpGODNs, Type C CpG ODNs induce strong B cell stimulation.

Exemplary stimulatory CpG ODNs comprise, but are not limited to, ODN1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN2395, ODN M362 (all InvivoGen). The present invention also encompassesany humanized version of the preceding CpG ODNs. In one preferredembodiment, compositions, methods, and devices of the present inventioncomprise ODN 1826 (the sequence of which from 5′ to 3′ istccatgacgttcctgacgtt, wherein CpG elements are bolded, SEQ ID NO: 10).

Neutral, or control, CpG ODNs that do not stimulate TLR9 are encompassedby the present invention. These ODNs comprise the same sequence as theirstimulatory counterparts but contain GpC dinucleotides in place of CpGdinucleotides.

Exemplary neutral, or control, CpG ODNs encompassed by the presentinvention comprise, but are not limited to, ODN 1585 control, ODN 1668control, ODN 1826 control, ODN 2006 control, ODN 2216 control, ODN 2336control, ODN 2395 control, ODN M362 control (all InvivoGen). The presentinvention also encompasses any humanized version of the preceding CpGODNs.

Suppressive CpG ODNs that inhibit TLR9 are encompassed by the presentinvention. Exemplary potent inhibitory sequences are (TTAGGG)₄ (ODNTTAGGG, InvivoGen), found in mammalian telomeres and ODN 2088(InvivoGen), derived from a murine stimulatory CpG ODN by replacement of3 bases. Suppressive ODNs disrupt the colocalization of CpG ODNs withTLR9 in endosomal vesicles without affecting cellular binding anduptake. Suppressive CpG ODNs encompassed by the present invention areused to fine-tune, attenuate, reverse, or oppose the action of astimulatory CpG-ODN. Alternatively, or in addition, compositions,methods, or devices of the present invention comprising suppressive CpGODNs are used to treat autoimmune conditions or prevent immune responsesfollowing transplant procedures.

Cancer Antigens

Compositions, methods, and devices of the present invention comprisecancer antigens with means to vaccinate and/or provide protectiveimmunity to a subject to whom such a device was administered. Cancerantigens are used alone or in combination with GM-CSF, CpG-ODNsequences, or immunomodulators. Moreover, cancer antigens are usedsimultaneously or sequentially with GM-CSF, CpG-ODN sequences, orimmunomodulators.

Exemplary cancer antigens encompassed by the compositions, methods, anddevices of the present invention include, but are not limited to, tumorlysates extracted from biopsies, irradiated tumor cells, MAGE series ofantigens (MAGE-1 is an example), MART-1/melana, tyrosinase, ganglioside,gp100, GD-2, 0-acetylated GD-3, GM-2, MUC-1, Sosl, Protein kinaseC-binding protein, Reverse transcriptase protein, AKAP protein, VRK1,KIAA1735, T7-1, T11-3, T11-9, Homo Sapiens telomerase ferment (hTRT),Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1),(PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovariancarcinoma antigen CAl25 (1A1-3B) (KIAA0049), MUCIN 1 (TUMOR-ASSOCIATEDMUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN),(PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANEANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM),(BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3), CTCL tumor antigen se1-1,CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigense20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumorantigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membraneantigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi'ssarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7),MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10), MAGE-B2 ANTIGEN (DAM6),MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigenNY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associatedsurface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associatedbrain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplasticneuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2),Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGENCO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint2, Squamous cell carcinoma antigen recognized by T cell, Serologicallydefined colon cancer antigen 1, Serologically defined breast cancerantigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16,Chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA72-4, CA 195, Carcinoembryonic antigen (CEA).

Immunomodulators

Compositions, methods, and devices of the present invention compriseimmunomodulators including, but not limited to, TLR ligands, growthfactors, and products of dying cells, e.g. heat shock proteins, withmeans to stimulate dendritic cell activation. Immunomodulators are usedalone or in combination with GM-CSF, CpG-ODN sequences, or cancerantigens Immunomodulators are used simultaneously or sequentially withGM-CSF, CpG-ODN sequences, or cancer antigens.

All known TLR ligands found either on a cell surface or an internalcellular compartment are encompassed by the compositions, methods, anddevices of the present invention. Exemplary TLR ligands include, but arenot limited to, triacyl lipoproteins (TLR1); lipoproteins, gram positivepeptidoglycan, lipteichoic acids, fungi, and viral glycoproteins (TLR2);double-stranded RNA, poly I:C (TLR 3); lipopolysaccaride, viralglycoproteins (TLR 4); flagellin (TLR5); diacyl lipoproteins (TLR6);small synthetic compounds, single-stranded RNA (TLR7 and TLR 8);unmethylated CpG DNA (TLR9); Profilin (TLR11). Also included as TRLligands are host molecules like fibronectin and heat shock proteins(HSPs). Host TLR ligands are also encompassed by the present invention.The role of TLRs in innate immunity and the signaling molecules used toactivate and inhibit them are known in the art (for a review, see HolgerK. Frank B., Hessel E., and Coffman R L. Therapeutic targeting of innateimmunity with Toll-like receptor agonists and antagonists. NatureMedicine 13, 552-559 (2007), herein incorporated by reference).

All known growth factors are encompassed by the compositions, methods,and devices of the present invention. Exemplary growth factors include,but are not limited to, transforming growth factor beta (TGF-β),granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), nerve growth factor (NGF),neurotrophins, Platelet-derived growth factor (PDGF), erythropoietin(EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiationfactor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basicfibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF),hepatocyte growth factor (HGF). The present invention encompassescytokines as well as growth factors for stimulating dendritic cellactivation. Exemplary cytokines include, but are not limited to, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 IL-15, IL-17, IL-18,TNF-α, IFN-γ, and IFN-α.

Indications of cell death and products of dying cells stimulatedendritic cell activation. As such, all products of dying cells areencompassed by the compositions, methods, and devices of the presentinvention. Exemplary cell death products include, but are not limitedto, any intracellular feature of a cell such as organelles, vesicles,cytoskeletal elements, proteins, DNA, and RNA. Of particular interestare heat shock proteins expressed when a cell is under stress and whichare released upon cell death. Exemplary heat shock proteins include, butare not limited to, Hsp10, Hsp20, Hsp27, Hsp33, Hsp40, Hsp60, Hsp70,Hsp71, Hsp72, Grp78, Hsx70, Hsp84, Hsp90, Grp94, Hsp100, Hsp104, Hsp110.

Microenvironments and Vaccine Efficiency

The devices/scaffold described herein represent an infection-mimickingmicroenvironment. Each device constitutes a factory thatattracts/accepts, educates/stimulates and sends forth to surroundingbodily tissues activated dendritic cells that are capable ofstimulating/enhancing an immune response to a particular antigen.Specifically, the scaffold devices are implanted or coated withpathogenic molecules to mimic and infectious microenvironment to furtheractivate the dendritic cell response.

Appropriately mimicking aspects of infection with material systemsdramatically impacts tumor progression when applied as cancer vaccinesby continuously recruiting, activating and homing DCs to LNs. The firstPLG vaccine, using GM-CSF alone, led to a batch process where host DCswere recruited by GM-CSF to reside at a site of tumor antigenpresentation, and were trapped until GM-CSF levels fell and the cellscould become activated and disperse (see U.S. Ser. No. 11/638,796;herein incorporated by reference). Temporal variation of the localGM-CSF concentration allowed control over the number of recruited DCs,and the timing of their activation and dispersement. Although the bestGM-CSF-based vaccine was able to confer protective immunity in nearly aquarter of the animals tested, approximately 26% of the recruited DCswere activated (240,000 DCs) and approximately 6% of DCs dispersed tothe LNs. High levels of GM-CSF recruited large numbers of DC, but alsolimited DC activation, leaving potentially therapeutic DCs entrappedwithin scaffolds. These results motivated the development of an improvedsystem that mimicked bacterial infection by locally presenting CpG-ODNsas an overriding ‘danger signal’, that opposed GM-CSF inhibition of DCactivation and dispersement. These devices described herein representsignificant advances by mediating increased and continuous egress ofDCs.

CpG-ODN molecules were condensed with PEI to not only promote ODN uptakeinto DCs and localization to its TLR-9 receptor (FIGS. 3A-D), but alsoto electrostatically immobilize it in PLG matrices to be presentedsimultaneously with tumor antigens (FIGS. 6A-C). In vitro resultsindicated that PEI-CpG-ODN condensates can decondense within DCs andstimulate TLR signaling that promoted DC activation and dispersementtoward the lymph node derived chemokine, CCL19, in the presence ofinhibitory levels of GM-CSF (500 ng/ml).

In vivo, appropriately designed infection-mimics mediated a continuousprocess that shuttled DCs through an infectious-like microenvironmentvia recruitment with GM-CSF, followed by immediate activation ofresident DCS via condensed CpG-ODN presentation, and subsequent release.An in vivo screen of the dose effects of combined CpG-ODN deliveryrevealed differential effects on DC activation, with an unusualdecoupling of CCR7 and MHCII expression, at high CpG-ODN (>50 μg) andGM-CSF (>1 μg) doses, whereas optimal CpG-ODN doses (10-25 μg) inducedsignificant DC activation (44%, and 1.5×10⁶ cells) even when opposed byhigh GM-CSF levels (3 μg, in vivo). Therefore, optimal CpG-ODNpresentation can activate large numbers of DCs recruited by strongGM-CSF pulses in situ, and these numbers exceed the numbers oftenprogrammed and transplanted in ex vivo protocols (FIGS. 7A-B).

This DC programming process proved to be continuous as DCs were shuttledthrough an infectious-like microenvironment via recruitment with intensepulses of GM-CSF, followed by the subsequent programming and release ofresident DCS via condensed CpG-ODN stimulation. The percentage of DCsthat homed to the LNs approximately doubled from 6% to 13% (U.S. Ser.No. 11/638,796 and FIGS. 8A-D), which corresponded to 180,000 programmedDCs (˜4-fold enhancement compared to devices without CpG-ODN) beingdispersed to the lymph nodes, with infection-mimics (FIGS. 7A-B and8A-D). Strikingly, the lymph nodes in this condition were markedlyenlarged (FIGS. 8A-D) and loaded with large numbers of DCs at sacrifice,supporting the conclusion that an infection-mimic was created in thoseanimals.

The ability of these infectious-material systems to continuously controlDC trafficking and activation translated to a regulation over theefficacy of the cancer vaccine. As the numbers of material-resident,activated DCs that were programmed and dispersed to the lymph nodesincreased, the efficacy increased from 0 to 23 and finally 50%. HostT-cells mediated the immune protection, and a clear relation between thenumbers of CD-4 and CD-8 lymphocytes (˜50% increase due to infectionmimicking) in the tumors that did form (FIGS. 10A-B) and vaccineefficacy was found. These results are qualitatively consistent with anex vivo vaccine developed using irradiated tumor cells engineered tosecrete GM-CSF, as that system was previously found to stimulate apotent, specific, and long-lasting anti-tumor immunity (Akira S, TakedaK, Kaisho T. Nature Immunol, 2, 675-80, 2001). In contrast, though, theinfection-mimicking material system programmed DCs in situ, and bypassedall ex vivo cell manipulation and transplantation, and provided tightcontrol over the number of DCs recruited, activated and dispersed to thelymph nodes (LNs).

These results indicate the value of finely controlling cell behavior andprogramming in situ. The mechanism behind vaccine efficacy in thesestudies was clearly the appropriate control over the number and timingof DC mobilization and programming. Infection-mimics are a useful toolfor the development of vaccines with means to create immunity againstotherwise lethal infection, cancers and autoimmunity.

Scaffold Compositions and Architecture

Components of the scaffolds are organized in a variety of geometricshapes (e.g., discs, beads, pellets), niches, planar layers (e.g., thinsheets). For example, discs of about 0.1-200 millimeters in diameter,e.g., 5, 10, 20, 40, 50 millimeters are implanted subcutaneously. Thedisc may have a thickness of 0.1 to 10 millimeters, e.g., 1, 2, 5millimeters. The discs are readily compressed or lyophilized foradministration to a patient. An exemplary disc for subcutaneousadministration has the following dimensions: 8 millimeters in diameterand 1 millimeter in thickness. Multicomponent scaffolds are optionallyconstructed in concentric layers each of which is characterized bydifferent physical qualities (% polymer, % crosslinking of polymer,chemical composition of scaffold, pore size, porosity, and porearchitecture, stiffness, toughness, ductility, viscoelasticity, and orcomposition of bioactive substances such as growth factors,homing/migration factors, differentiation factors. Each niche has aspecific effect on a cell population, e.g., promoting or inhibiting aspecific cellular function, proliferation, differentiation, elaborationof secreted factors or enzymes, or migration. Cells incubated in thescaffold are educated and induced to migrate out of the scaffold todirectly affect a target tissue, e.g., and injured tissue site. Forexample, stromal vascular cells and smooth muscle cells are useful insheet-like structures are used for repair of vessel-like structures suchas blood vessels or layers of the body cavity. For example, suchstructures are used to repair abdominal wall injuries or defects such asgastroschisis. Similarly, sheet-like scaffolds seeded with dermal stemcells and/or keratinocytes are used in bandages or wound dressings forregeneration of dermal tissue. The device is placed or transplanted onor next to a target tissue, in a protected location in the body, next toblood vessels, or outside the body as in the case of an external wounddressing. Devices are introduced into or onto a bodily tissue using avariety of known methods and tools, e.g., spoon, tweezers or graspers,hypodermic needle, endoscopic manipulator, endo- ortrans-vascular-catheter, stereotaxic needle, snake device,organ-surface-crawling robot (United States Patent Application20050154376; Ota et al., 2006, Innovations 1:227-231), minimallyinvasive surgical devices, surgical implantation tools, and transdermalpatches. Devices can also be assembled in place, for example bysequentially injecting or inserting matrix materials. Scaffold devicesare optionally recharged with cells or with bioactive compounds, e.g.,by sequential injection or spraying of substances such as growth factorsor differentiation factors.

A scaffold or scaffold device is the physical structure upon which orinto which cells associate or attach, and a scaffold composition is thematerial from which the structure is made. For example, scaffoldcompositions include biodegradable or permanent materials such as thoselisted below. The mechanical characteristics of the scaffold varyaccording to the application or tissue type for which regeneration issought. It is biodegradable (e.g., collagen, alginates, polysaccharides,polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA),or poly(lactide-co-glycolide) (PLGA), poly lactic-coglycolic acid, orpermanent (e.g., silk). In the case of biodegradable structures, thecomposition is degraded by physical or chemical action, e.g., level ofhydration, heat or ion exchange or by cellular action, e.g., elaborationof enzyme, peptides, or other compounds by nearby or resident cells. Theconsistency varies from a soft/pliable (e.g., a gel) to glassy, rubbery,brittle, tough, elastic, stiff. The structures contain pores, which arenanoporous, microporous, or macroporous, and the pattern of the pores isoptionally homogeneous, heterogeneous, aligned, repeating, or random.

Alginates are versatile polysaccharide based polymers that may beformulated for specific applications by controlling the molecularweight, rate of degradation and method of scaffold formation. Couplingreactions can be used to covalently attach bioactive epitopes, such asthe cell adhesion sequence RGD to the polymer backbone. Alginatepolymers are formed into a variety of scaffold types. Injectablehydrogels can be formed from low MW alginate solutions upon addition ofa cross-linking agents, such as calcium ions, while macroporousscaffolds are formed by lyophilization of high MW alginate discs.Differences in scaffold formulation control the kinetics of scaffolddegradation. Release rates of morphogens or other bioactive substancesfrom alginate scaffolds is controlled by scaffold formulation to presentmorphogens in a spatially and temporally controlled manner. Thiscontrolled release not only eliminates systemic side effects and theneed for multiple injections, but can be used to create amicroenvironment that activates host cells at the implant site andtransplanted cells seeded onto a scaffold.

The scaffold comprises a biocompatible polymer matrix that is optionallybiodegradable in whole or in part. A hydrogel is one example of asuitable polymer matrix material. Examples of materials which can formhydrogels include polylactic acid, polyglycolic acid, PLGA polymers,alginates and alginate derivatives, gelatin, collagen, agarose, naturaland synthetic polysaccharides, polyamino acids such as polypeptidesparticularly poly(lysine), polyesters such as polyhydroxybutyrate andpoly-epsilon.-caprolactone, polyanhydrides; polyphosphazines, poly(vinylalcohols), poly(alkylene oxides) particularly poly(ethylene oxides),poly(allylamines)(PAM), poly(acrylates), modified styrene polymers suchas poly(4-aminomethylstyrene), pluronic polyols, polyoxamers,poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above,including graft copolymers.

The scaffolds are fabricated from a variety of synthetic polymers andnaturally-occurring polymers such as, but not limited to, collagen,fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferredmaterial for the hydrogel is alginate or modified alginate material.Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (Munits) and a L-guluronic acid (G units) monomers, which can vary inproportion and sequential distribution along the polymer chain. Alginatepolysaccharides are polyelectrolyte systems which have a strong affinityfor divalent cations (e.g. Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogelswhen exposed to these molecules. See Martinsen A., et al., Biotech. &Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginatehydrogels are useful for dental applications, wound dressingschondrocyte transplantation and as a matrix for other cell types.

An exemplary device utilizes an alginate or other polysaccharide of arelatively low molecular weight, preferably of size which, afterdissolution, is at the renal threshold for clearance by humans, e.g.,the alginate or polysaccharide is reduced to a molecular weight of 1000to 80,000 daltons. Preferably, the molecular mass is 1000 to 60,000daltons, particularly preferably 1000 to 50,000 daltons. It is alsouseful to use an alginate material of high guluronate content since theguluronate units, as opposed to the mannuronate units, provide sites forionic crosslinking through divalent cations to gel the polymer. U.S.Pat. No. 6,642,363, incorporated herein by reference discloses methodsfor making and using polymers containing polysaccharides such asalginates or modified alginates that are particularly useful for celltransplantation and tissue engineering applications.

Useful polysaccharides other than alginates include agarose andmicrobial polysaccharides such as those listed in the table below.

Polysaccharide Scaffold Compositions Polymers^(a) Structure FungalPullulan (N) 1,4-; 1,6-α-D-Glucan Scleroglucan (N) 1,3; 1,6-α-D-GlucanChitin (N) 1,4-β-D-Acetyl Glucosamine Chitosan (C)1,4-β.-D-N-Glucosamine Elsinan (N) 1,4-; 1,3-α-D-Glucan BacterialXanthan gum (A) 1,4-β.-D-Glucan with D-mannose; D-glucuronic Acid asside groups Curdlan (N) 1,3-β.-D-Glucan (with branching) Dextran (N)1,6-α-D-Glucan with some 1,2; 1,3-; 1,4-α-linkages Gellan (A)1,4-β.-D-Glucan with rhamose, D-glucuronic acid Levan (N)2,6-β-D-Fructan with some β-2,1-branching Emulsan (A)Lipoheteropolysaccharide Cellulose (N) 1,4-β-D-Glucan ^(a)N—neutral, A =anionic and C = cationic.

The scaffolds of the invention are porous or non-porous. For example,the scaffolds are nanoporous having a diameter of less than about 10 nm;microporous wherein the diameter of the pores are preferably in therange of about 100 nm-20 μm; or macroporous wherein the diameter of thepores are greater than about 20 μm, more preferably greater than about100 μm and even more preferably greater than about 400 μm. In oneexample, the scaffold is macroporous with aligned pores of about 400-500μm in diameter. The preparation of polymer matrices having the desiredpore sizes and pore alignments are described in the Examples. Othermethods of preparing porous hydrogel products are known in the art.(U.S. Pat. No. 6,511,650 incorporated herein by reference).

Bioactive Compositions

The device includes one or more bioactive compositions. Bioactivecompositions are purified naturally-occurring, synthetically produced,or recombinant compounds, e.g., polypeptides, nucleic acids, smallmolecules, or other agents. For example, the compositions includeGM-CSF, CpG-ODN, and tumor antigens or other antigens. The compositionsdescribed herein are purified. Purified compounds are at least 60% byweight (dry weight) the compound of interest. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Purity ismeasured by any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Coupling of the polypeptides to the polymer matrix is accomplished usingsynthetic methods known to one of ordinary skill in the art. Approachesto coupling of peptides to polymers are discussed in Hirano and Mooney,Advanced Materials, p. 17-25 (2004). Other useful bonding chemistriesinclude those discussed in Hermanson, Bioconjugate Techniques, p.152-185 (1996), particularly by use of carbodiimide couplers, DCC andDIC (Woodward's Reagent K). Polypeptides contain a terminal amine groupfor such carbodiimide bonding. The amide bond formation is preferablycatalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), whichis a water soluble enzyme commonly used in peptide synthesis.

Control of Release Kinetics of Bioactive Compositions

The release profile of bioactive compositions such as GM-CSF iscontrolled using a number of different techniques, e.g., encapsulation,nature of attachment/association with the scaffold, porosity of thescaffold, and particle size of the bioactive compositions.

For example, GM-CSF is encapsulated as one means by which to incorporateGM-CSF into the scaffolds. GM-CSF was first encapsulated into PLGmicrospheres, and then these GM-CSF loaded microspheres were then in agas foaming process to develop macroporous PLG scaffolds. Theincorporation of GM-CSF into the microspheres causes the GM-CSF to bemore deeply embedded into the polymer, which causes the device tosustain the initial pulse of GM-CSF delivery over days 1-5. Otherincorporation methods are optionally used to alter or fine tune theduration of the GM-CSF pulse as desired, which would in turn change thekinetics of DC recruitment. For example, foaming PLG particles mixedwith lyophilized GM-CSF results in GM-CSF that is associated more withthe surface of the polymer scaffold, and the protein diffuses morequickly.

Alternative methods for scaffold fabrication that modify releasekinetics include modifying the physical structure of the scaffoldspores, thereby leading to different degradation times and releasekinetics (change pore size or total porosity as a percentage of volume),e.g., as described in Riddle et al., Role of poly(lactide-co-glycolide)particle size on gas-foamed scaffolds. J Biomater Sci Polym Ed. 2004;15(12):1561-70. Another way to alter release kinetics is to modify thecomposition, i.e., the raw materials from which the scaffold is made,thereby altering the release properties. For example, differentpolymers, e.g. alginate, PLA, PGA, or using PLGA are used. Also, use ofthe polymers with different ratios of glycolic and lactic acid) leads todifferent release profiles. For example, a variety of PLGs, differing incomposition (lactide to glycolide ratio) and molecular weight are usedto prepare microspheres (5-50 μm) using known double emulsion(water/oil/water) process, followed by preparation of scaffolds usingparticulate PLG and PLG microspheres using gas foaming/particulateleaching techniques (Ennett et al., Temporally regulated delivery ofVEGF in vitro and in vivo. J Biomed Mater Res A. 2006 October; 79(1).Another technique involves incorporating the protein into differentcompartments (e.g., encapsulating proteins PLG microspheres or simplemixing and lyophilizing with the polymer before foaming).

Charging and/or Recharging the Device

A bioactive composition such as GM-CSF is incorporated within differentlayers/compartments of the device, thereby allowing multiple pulses ofGM-CSF to be delivered. Each pulse charges (or recharges) the devicewith an influx of DCs. Scaffolds are fabricated using a variety ofmethods to create multiple pulses of GM-CSF (or other bioactive agents).For example, such devices are made by incorporating the protein intodifferent compartments (e.g encapsulating proteins PLG microspheres orsimple mixing and lyophilizing with the polymer before foaming) therebycreating 2 or more distinct release profiles (i.e. pulses) of theprotein (e.g., as described in Richardson et al., Polymeric system fordual growth factor delivery. Nat Biotechnol. 2001 November; 19(11)).

Alternatively, the protein is encapsulated in fast degrading PLGmicrospheres (e.g. low MW, 50:50 ratio) and slow degrading PLGmicrospheres (high MW, 85:15 ratio). Then these microspheres are mixedtogether to be used later to fabricate the scaffolds. Therefore, theprotein is encapsulated in both fast a degrading polymer and a slowdegrading polymer, thereby resulting in at least 2 distinct releaseskinetics and pulses of delivery. This method is utilized to create 3, 4,5, or more different kinds of microspheres, the ratiometriccharacteristics of which differ, thereby leading to 3, 4, 5 or morepulses of release of the bioactive composition such as GM-CSF.

Another approach to making a device that delivers more than one pulse isto fabricate a layered scaffold. Layered scaffolds are made bycompression molding on different scaffold formulations with another. Forexample, the raw materials (sucrose+PLG1+Protein) is compressed in amold and a slightly varied formulation (sucrose+PLG2+Protein) is alsocompressed in a mold. Then these two layers are compressed together andthen foamed, resulting in a bilayered scaffold with distinct spatialcontrol of the concentration of the protein, e.g., as described in Chenet al., Pharm Res. Spatio-temporal VEGF and PDGF delivery patterns bloodvessel formation and maturation. 2007 February; 24(2):258-64).

Device Construction

The scaffold structure is constructed out of a number of differentrigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline,or fluid compositions such as peptide polymers, polysaccharides,synthetic polymers, hydrogel materials, ceramics (e.g., calciumphosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans,metals and metal alloys. The compositions are assembled into cellscaffold structures using methods known in the art, e.g., injectionmolding, lyophillization of preformed structures, printing,self-assembly, phase inversion, solvent casting, melt processing, gasfoaming, fiber forming/processing, particulate leaching or a combinationthereof. The assembled devices are then implanted or administered to thebody of an individual to be treated.

The device is assembled in vivo in several ways. The scaffold is madefrom a gelling material, which is introduced into the body in itsungelled form where it gels in situ. Exemplary methods of deliveringdevice components to a site at which assembly occurs include injectionthrough a needle or other extrusion tool, spraying, painting, or methodsof deposit at a tissue site, e.g., delivery using an application deviceinserted through a cannula. In one example, the ungelled or unformedscaffold material is mixed with bioactive substances and cells prior tointroduction into the body or while it is introduced. The resultant invivo/in situ assembled scaffold contains a mixture of these substancesand cells.

In situ assembly of the scaffold occurs as a result of spontaneousassociation of polymers or from synergistically or chemically catalyzedpolymerization. Synergistic or chemical catalysis is initiated by anumber of endogenous factors or conditions at or near the assembly site,e.g., body temperature, ions or pH in the body, or by exogenous factorsor conditions supplied by the operator to the assembly site, e.g.,photons, heat, electrical, sound, or other radiation directed at theungelled material after it has been introduced. The energy is directedat the scaffold material by a radiation beam or through a heat or lightconductor, such as a wire or fiber optic cable or an ultrasonictransducer. Alternatively, a shear-thinning material, such as anampliphile, is used which re-cross links after the shear force exertedupon it, for example by its passage through a needle, has been relieved.

Suitable hydrogels for both in vivo and ex vivo assembly of scaffolddevices are well known in the art and described, e.g., in Lee et al.,2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach toself-assembly assembly is described, e.g., in Hartgerink et al., 2002,Proc. Natl. Acad. Sci. U.S.A 99:5133-5138. A method for reversiblegellation following shear thinning is exemplified in Lee et al., 2003,Adv. Mat. 15:1828-1832.

A multiple compartment device is assembled in vivo by applyingsequential layers of similarly or differentially doped gel or otherscaffold material to the target site. For example, the device is formedby sequentially injecting the next, inner layer into the center of thepreviously injected material using a needle, forming concentricspheroids. Non-concentric compartments are formed by injecting materialinto different locations in a previously injected layer. A multi-headedinjection device extrudes compartments in parallel and simultaneously.The layers are made of similar or different scaffolding compositionsdifferentially doped with bioactive substances and different cell types.Alternatively, compartments self-organize based on theirhydro-philic/phobic characteristics or on secondary interactions withineach compartment.

Compartmentalized Device

In certain situations, a device containing compartments with distinctchemical and/or physical properties is useful. A compartmentalizeddevice is designed and fabricated using different compositions orconcentrations of compositions for each compartment.

Alternatively, the compartments are fabricated individually, and thenadhered to each other (e.g., a “sandwich” with an inner compartmentsurrounded on one or all sides with the second compartment). This latterconstruction approach is accomplished using the intrinsic adhesivenessof each layer for the other, diffusion and interpenetration of polymerchains in each layer, polymerization or cross-linking of the secondlayer to the first, use of an adhesive (e.g., fibrin glue), or physicalentrapment of one compartment in the other. The compartmentsself-assemble and interface appropriately, either in vitro or in vivo,depending on the presence of appropriate precursors (e.g., temperaturesensitive oligopeptides, ionic strength sensitive oligopeptides, blockpolymers, cross-linkers and polymer chains (or combinations thereof),and precursors containing cell adhesion molecules that allowcell-controlled assembly).

Alternatively, the compartmentalized device is formed using a printingtechnology. Successive layers of a scaffold precursor doped withbioactive substances is placed on a substrate then cross linked, forexample by self-assembling chemistries. When the cross linking iscontrolled by chemical-, photo- or heat-catalyzed polymerization, thethickness and pattern of each layer is controlled by a masque, allowingcomplex three dimensional patterns to be built up when un-cross-linkedprecursor material is washed away after each catalyzation. (WT Brinkmanet al., Photo-cross-linking of type 1 collagen gels in the presence ofsmooth muscle cells: mechanical properties, cell viability, andfunction. Biomacromolecules, 2003 July-August; 4(4): 890-895.; W. Ryu etal., The construction of three-dimensional micro-fluidic scaffolds ofbiodegradable polymers by solvent vapor based bonding of micro-moldedlayers. Biomaterials, 2007 February; 28(6): 1174-1184; Wright, Paul K.(2001). 21st Century manufacturing. New Jersey: Prentice-Hall Inc.)Complex, multi-compartment layers are also built up using an inkjetdevice which “paints” different doped-scaffold precursors on differentareas of the substrate. Julie Phillippi (Carnegie Mellon University)presentation at the annual meeting of the American Society for CellBiology on Dec. 10, 2006; Print me a heart and a set of arteries,Aldhouse P., New Scientist 13 Apr. 2006 Issue 2547 p 19.; Replacementorgans, hot off the press, C. Choi, New Scientist, 25 Jan. 2003, v2379.These layers are built-up into complex, three dimensional compartments.The device is also built using any of the following methods: JettedPhotopolymer, Selective Laser Sintering, Laminated Object Manufacturing,Fused Deposition Modeling, Single Jet Inkjet, Three DimensionalPrinting, or Laminated Object Manufacturing.

The release profiles of bioactive substances from scaffold devices iscontrolled by both factor diffusion and polymer degradation, the dose ofthe factor loaded in the system, and the composition of the polymer.Similarly, the range of action (tissue distribution) and duration ofaction, or spatiotemporal gradients of the released factors areregulated by these variables. The diffusion and degradation of thefactors in the tissue of interest is optionally regulated by chemicallymodifying the factors (e.g., PEGylating growth factors). In both cases,the time frame of release determines the time over which effective celldelivery by the device is desired.

The bioactive substances are added to the scaffold compositions usingknown methods including surface absorption, physical immobilization,e.g., using a phase change to entrap the substance in the scaffoldmaterial. For example, a growth factor is mixed with the scaffoldcomposition while it is in an aqueous or liquid phase, and after achange in environmental conditions (e.g., pH, temperature, ionconcentration), the liquid gels or solidifies thereby entrapping thebioactive substance. Alternatively, covalent coupling, e.g., usingalkylating or acylating agents, is used to provide a stable, long termpresentation of a bioactive substance on the scaffold in a definedconformation. Exemplary reagents for covalent coupling of suchsubstances are provided in the table below.

Methods to Covalently Couple Peptides/Proteins to Polymers

Functional Group Coupling reagents Reacting groups on of Polymer andcross-linker proteins/peptides —OH Cyanogen bromide (CNBr) —NH₂ Cyanuricchloride 4-(4,6-Dimethoxy-1,3,5- triazin-2-yl)-4-methyl- morpholiniumchloride (DMT-MM) —NH₂ Diisocyanate compounds —NH₂ Diisothoncyanatecompounds —OH Glutaraldehyde Succinic anhydride —NH₂ Nitrous Acid —NH₂Hydrazine + nitrous acid —SH —Ph—OH —NH₂ Carbodiimide compounds (e.g.,—COOH EDC, DCC)[a] DMT-MM —COOH Thionyl chloride —NH₂N-hydroxysuccinimide N-hydroxysulfosuccinimide + EDC —SH Disulfidecompound —SH [a]EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride; DCC: dicyclohexylcarbodiimide

Bioactive substances suitable for use in the present invention include,but are not limited to: interferons, interleukins, chemokines,cytokines, colony stimulating factors, chemotactic factors,granulocyte/macrophage colony stimulating factor (GMCSF). Splicevariants of any of the above mentioned proteins, and small moleculeagonists or antagonists thereof that may be used advantageously toactivate dendritic cells are also contemplated herein.

Examples of cytokines as mentioned above include, but are not limited toIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN),IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40ligand.

Scaffolds of the invention optionally comprise at least one non-viralgene therapy vector such that either the transplanted cells or hostcells in the vicinity of the implant would take up and express gene thatlead to local availability of the desired factor for a desirable timeframe. Such non-viral vectors include, but are not limited to, cationiclipids, polymers, targeting proteins, and calcium phosphate.

Scaffold Fabrication.

A 85:15, 120 kD copolymer of D,L-lactide and glycolide (PLG) (Alkermes,Cambridge, Mass.) was utilized in a gas-foaming process to formscaffolds with open, interconnected pores (Cohen S., Yoshioka T.,Lucarelli, M., Hwang L. H., and Langer R. Pharm. Res. 8, 713-720 (1991);herein incorporated by reference). PLG microspheres encapsulating GM-CSFwere made using standard double emulsion (Harris, L. D., Kim, B. S., andMooney, D. J. J. Biomed. Mater. Res. 42, 396-402 (1998); hereinincorporated by reference). 16 mg of PLG microspheres were then mixedwith 150 mg of the porogens, NaCl or sucrose (sieved to a particle sizebetween 250 μm and 425 μm), and compression molded. The resulting discwas allowed to equilibrate within a high-pressure CO₂ environment, and arapid reduction in pressure causes the polymer particles to expand andfuse into an interconnected structure. The NaCl was leached from thescaffolds by immersion in water yielding scaffolds that were 90% porous.To incorporate tumor lysates into PLG scaffolds, biopsies of B16-F10tumors, that had grown subcutaneously in the backs of C57BL/6J mice(Jackson Laboratory, Bar Harbor Me.), were digested in collagenase (250U/ml) (Worthington, Lakewood, N.J.) and suspended at a concentrationequivalent to 10⁷ cells per ml after filtration through 40 μm cellstrainers. The tumor cell suspension was subjected to 4 cycles of rapidfreeze in liquid nitrogen and thaw (37° C.) and then centrifuged at 400rpm for 10 min. The supernatant (1 ml) containing tumor lysates wascollected and lyophilized with the PLG microspheres and the resultingmixture was used to make PLG scaffold-based cancer vaccines. Toincorporate CpG-ODNs into PLG scaffolds, PEI-CpG-ODN condensatesolutions were vortexed with 60 μl of 50% (wt/vol) sucrose solution,lyophilized and mixed with dry sucrose to a final weight of 150 mg. Thesucrose containing PEI-CpG-ODN condensate was then mixed with blank,GM-CSF and/or tumor lysate loaded PLG microspheres to make PLG cancervaccines.

Scaffold compositions of the present invention comprise GM-CSF andCpG-ODN sequences. A range of concentrations of each element arecontemplated. In a preferred embodiment, the scaffold compositioncomprises PLG. With respect to GM-CSF, per 40 mg polymeric scaffoldcomposition, 0-100 μg of GM-CSF polypeptide is incorporated into orcoated onto the scaffold composition. Alternatively, doses comprising0-50 μg, 0-25 μg, 0-10 μg, 0-5 μg, and 0-3 μg of GM-CSF are incorporatedinto the scaffold composition. In a preferred embodiment, 0-3 μg ofGM-CSF are incorporated into the scaffold composition. With respect toCpG-ODN sequences, or PEI-CpG-ODN condensates, per 40 mg polymericscaffold composition, 0-1000 μg of PEI-CpG-ODN is incorporated into orcoated onto the scaffold composition. Alternatively, doses comprising0-500 μg, 0-250 μg, 0-100 μg, 0-50 μg, 0-25 μg, 0-10 μg, and 0-5 μg ofPEI-CpG-ODN are incorporated into the scaffold composition. In apreferred embodiment, 0-50 μg of PEI-CpG-ODN are incorporated into thescaffold composition.

CpG-ODN Incorporation and In Vitro Release Studies

To determine the incorporation efficiency of CpG-ODN incorporation, PLGscaffolds were prepared with 50 μg of CpG-ODN and digested in 1 ml ofchloroform (Sigma Aldrich, and washed with 2 mls of aqueous buffer. Theaqueous phase was isolated and the amount of CpG-ODN incorporated wasdetermined by absorbance readings (260/280 and 260/230 ratios calculatedat 0.2 mm pathlength) using a Nanodrop instrument, ND1000 (Nanodroptechnologies, Wilmington, Del.). Similarly, to determine CpG-ODN releasekinetics CpG-ODN loaded scaffolds were placed in 1 ml of PhosphateBuffer Solution (PBS) in an incubator (37° C.). At various timepoints,the PBS release media was collected and replaced with fresh media. Thetotal amount of CpG-ODN incorporated into PLG scaffolds and releasedinto PBS over time was analyzed and recorded.

In Vitro DC Migration Assays and DC Activation

A DC line, JAWSII (ATCC, Manassas, Va.) was used for in vitroexperiments and was maintained in α-MEM (Invitrogen, Carlsbad, Calif.)supplemented with 20% FBS (Invitrogen, Carlsbad, Calif.) and 5 ng/ml ofGM-CSF. To determine the in vitro effects of CpG-rich oligonucleotides(CpG-ODN) on DC activation, JAWSII cells were cultured with 5 μg/ml ofCpG-ODN 1826, 5′-tcc atg acg ttc ctg acg tt-3′, (SEQ ID NO: 10;Invivogen, San Diego, Calif.) for 24 hours, and in the presence of 0, 50or 500 ng/ml GM-CSF for 12 hours. To assess the effects of condensingCpG-ODN on DC activation, CpG ODN was condensed with PEI molecules bydropping ODN-1826 solutions into PEI solution, while vortexing themixture (Huang Y C, Riddle F, Rice K G, and Mooney D J. Hum Gene Ther.5, 609-17. (2005); herein incorporated by reference). The charge ratiobetween PEI and CpG-ODN (NH3+:PO4−) was kept constant at 7 duringcondensation. As a positive control for DC activation, DCs were alsocultured with the stimulatory factors, TNF-α (10 ng/ml) (Peprotech,Rocky Hill, N.J.) and LPS (10 ng/ml) (Sigma-Aldrich, St. Louis, Mo.).The DCs were then harvested and stained with primary antibodies (BDPharmingen, San Diego, Calif.): PE-conjugated CD86 (B7, costimulatorymolecule), FITC-conjugated CCR7, and FITC-conjugated MHCII. Cells wereanalyzed by FACS and gated according to positive FITC, and PE usingisotype controls, and the percentage of cells staining positive for eachsurface antigen was recorded.

Migration assays were performed with 6.5 mm transwell dishes (Costar,Cambridge, Mass.) with a pore size of 5 μm. To test whether CpG-ODNstimulation may affect DC chemotaxis towards CCL19 (Peprotech, RockyHill, N.J.) in the presence of GM-CSF, 5×10⁵ DCs stimulated with either5 μg/ml of CpG-ODN or PEI-CPG-ODN (Charge Ratio of 7), and 0, 50 and 500ng/ml GM-CSF were placed in the top wells and 300 ng/ml of CCL19 wasplaced in the bottom well. After 12 hours the cells that migrated intothe bottom wells of the chamber were harvested and counted using acoulter counter. Dispersement of DCs from PEI-CpG-ODN loaded PLGmatrices toward CCL19 was assessed by incorporating 5, 50 and 500 μg ofcondensates into PLG scaffolds (13 mm diameter, 2 mm thick that werequartered) seeded with 1×10⁶ DCs and fixed onto transwells using bovinecollagen (BD Biosciences, San Jose, Calif.). To test the effects of CpGstimulation in the presence of GM-CSF, 500 ng/ml of GM-CSF wassupplemented into the media of the top wells with scaffolds containing25 μg of PEI-CpG-ODN. At various timepoints, the cells that migratedinto the bottom wells of the chamber were harvested and counted using acoulter counter.

In Vivo DC Migration and Activation Assays

Blank scaffolds and scaffolds containing GM-CSF with or without 10 μgPEI-ODN control (5′-tcc atg agc ttc ctg agc tt-3′) (SEQ ID NO: 6) or 10μg PEI-CpG-ODN condensate loaded scaffolds were implanted intosubcutaneous pockets on the back of 7-9 week old male C57BL/6J mice. Forhistological examination scaffolds were excised and fixed in Z-fixsolution, embedded in paraffin, and stained with hematoxylin and eosin.To analyze DC recruitment, scaffolds were excised and the ingrown tissuewas digested into single cell suspensions using a collagenase solution(Worthingtion, 250 U/ml) that was agitated at 37° C. for 45 minutes. Thecell suspensions were then poured through a 40 μm cell strainer toisolate cells from scaffold particles and the cells were pelleted andwashed with cold PBS and counted using a Z2 coulter counter (BeckmanCoulter). The resultant cell populations were then stained with primaryantibodies (BD Pharmingen, San Diego, Calif.) conjugated to fluorescentmarkers to allow for analysis by flow cytometry. APC-conjugated CD11c(dendritic cell marker) and PE-conjugated CD86 (B7, costimulatorymolecule) stains were conducted for DC recruitment analysis, andAPC-conjugated CD11c, FITC-conjugated CCR7, and PE-conjugated MHCIIstains were conducted for DC programming analysis. Cells were gatedaccording to positive FITC, APC and PE using isotype controls, and thepercentage of cells staining positive for each surface antigen wasrecorded. To track in vivo DC emigration from scaffolds toward theinguinal lymph nodes, 250 μg of lyophilized fluoroscein isothiocyanate(FITC) (Molecular Probes, Carlsbad, Calif.) was incorporated intoscaffolds by mixing with PLG microspheres before scaffold processing,and FITC was also applied by incubating scaffolds with 330 ul of 3% FITCsolution for 30 min FITC painted scaffolds were then implantedsubcutaneously into the left flank of C57BL/6J mice and the inguinallymph nodes (LNs) were harvested at various time-points after scaffoldimplantation. Cell suspensions from LNs were prepared by digestion incollagenase for 30 min and pressing of the tissue through 70 μm cellstrainers, and examined for CD11c(+)FITC(+) cell numbers by flowcytometry.

Tumor Growth Assays

PLG scaffolds with melanoma tumor lysates and various dosages of GM-CSFand/or 10 μg PEI-CpG-ODN condensates were implanted subcutaneously intothe lower left flank of C57BL/6J mice. Animals were challenged 14 dayslater with a subcutaneous injection of 10⁵ B16-F10 melanoma cells (ATCC,Manassas, N.J.) in the back of the neck. Animals were monitored for theonset of tumor growth (approximately 1 mm³) and sacrificed for humanereasons when tumors grew to 20-25 mm (longest diameter). Forhistological examination, tumors were biopsied at days 20-25 afterinjection and fixed in Z-fix (Anatech, Battle Creek, Mich.) and stainedfor hematoxylin and eosin. To examine tumor tissue for T-cellinfiltration, immunoperoxidase staining was performed using theavidin-biotin-peroxidase Vectastain Elite ABC kit (Vector Laboratories).The primary antibodies used were GK 1.5 (CD4), and 53-6.72 (CD8) andstaining was developed using DAB+substrate chromogen (DAKO, Carpinteria,Calif.). Sections from tumor samples (n=3 or 4) were visualized at 40×and 100× with a Nikon light microscope (Indianapolis, Ind.) andpositively stained T-cells were counted manually. PLG cancer vaccineswere also compared to a common cell-based vaccine using B16-F10 melanomacells that were genetically modified to express GM-CSF, and subsequentlyirradiated (3500 rad) as described previously (Dranoff G., et al. Proc.Natl. Acad. Sci. USA. 90, 3539-3543(1993); herein incorporated byreference). The irradiated tumor cells (5×10⁵ cells) were then injectedsubcutaneously into C57BL/6J mice that were challenged 14 days laterwith 10⁵ B16-F10 melanoma cells.

Statistical Analysis

All values in the present study were expressed as mean±S.D. Thesignificant differences between the groups were analyzed by a Student'st test and a P value of less than 0.05 was considered significant.

Vaccine Device

The biocompatible scaffolds are useful as delivery vehicles for cancervaccines. The cancer vaccine stimulates an endogenous immune responseagainst cancer cells. Currently produced vaccines predominantly activatethe humoral immune system (i.e., the antibody dependent immuneresponse). Other vaccines currently in development are focused onactivating the cell-mediated immune system including cytotoxic Tlymphocytes which are capable of killing tumor cells. Cancer vaccinesgenerally enhance the presentation of cancer antigens to both antigenpresenting cells (e.g., macrophages and dendritic cells) and/or to otherimmune cells such as T cells, B cells, and NK cells. Although cancervaccines may take one of several forms, their purpose is to delivercancer antigens and/or cancer associated antigens to antigen presentingcells (APC) in order to facilitate the endogenous processing of suchantigens by APC and the ultimate presentation of antigen presentation onthe cell surface in the context of MHC class I molecules. One form ofcancer vaccine is a whole cell vaccine which is a preparation of cancercells which have been removed from a subject, treated ex vivo and thenreintroduced as whole cells in the subject. These treatments optionallyinvolve cytokine exposure to activate the cells, genetic manipulation tooverexpress cytokines from the cells, or priming with tumor specificantigens or cocktails of antigens, and expansion in culture. Dendriticcell vaccines activate antigen presenting cells directly, and theirproliferation, activation and migration to lymph nodes is regulated byscaffold compositions to enhance their ability to elicit an immuneresponse. Types of cancers to be treated include central nervous system(CNS) cancers, CNS Germ Cell tumor, lung cancer, Leukemia, MultipleMyeloma, Renal Cancer, Malignant Glioma, Medulloblastoma, and Melanoma.

For the purpose of eliciting an antigen-specific immune response, ascaffold device is implanted into a mammal. The device is tailored toactivate immune cells and prime the cells with a specific antigenthereby enhancing immune defenses and destruction of undesired tissuesand targeted microorganisms such as bacterial or viral pathogens. Thedevice attracts appropriate immune cells, such as macrophages, T cells,B cells, NK cells, and dendritic cells, by containing and/or releasingsignaling substances such as GM-CSF. These signaling substances areincorporated in the scaffold composition in such a way as to controltheir release spatially and temporally using the same techniques used tointegrate other bioactive compounds in the scaffold composition.

Once the immune cells are inside the device, the device programs theimmune cells to attack or cause other aspects of the immune system toattack undesired tissues (e.g., cancer, adipose deposits, orvirus-infected or otherwise diseased cells) or microorganisms Immunecell activation is accomplished by exposing the resident immune cells topreparations of target-specific compositions, e.g., ligands found on thesurface of the undesired tissues or organisms, such as cancer cellsurface markers, viral proteins, oligonucleotides, peptide sequences orother specific antigens. For example, useful cancer cell-specificantigens and other tissue or organism-specific proteins are listed inthe table below.

The device optionally contains multiple ligands or antigens in order tocreate a multivalent vaccine. The compositions are embedded in or coatedon the surface of one or more compartments of the scaffold compositionsuch that immune cells migrating through the device are exposed to thecompositions in their traverse through the device. Antigens or otherimmune stimulatory molecules are exposed or become exposed to the cellsas the scaffold composition degrades. The device may also containvaccine adjuvants that program the immune cells to recognize ligands andenhance antigen presentation. Exemplary vaccine adjuvants includechemokines/cytokines, CpG rich oligonucleotides. or antibodies that areexposed concurrently with target cell-specific antigens or ligands.

The device attracts immune cells to migrate into a scaffold where theyare educated in an antigen-specific manner and activated. The programmedimmune cells are then induced to egress towards lymph nodes in a numberof ways. The recruitment composition and deployment signal/composition,e.g., a lymph node migration inducing substance, is released in one ormore bursts, programmed by the method of incorporation and/or releasefrom the scaffold material, or controlled by the sequential degradationof scaffold compartments which contain the attractant. When a burstdissipates, the cells migrate away. Compartments containing repulsivesubstances are designed to degrade and release the repulsive substancein one or more bursts or steadily over time. Relative concentration ofthe repulsive substances cause the immune cells to migrate out of thedevice. Alternatively, cells which have been placed in or have migratedinto the device are programmed to release repulsive substances or tochange their own behavior. For example, localized gene therapy iscarried out by cell exposure to plasmid DNA attached to the scaffold.Useful repulsive substances include chemokines and cytokines.Alternatively, the device may cause immune cells to egress by degradingand releasing them.

Target disease states, stimulatory molecules and antigens useful invaccine device construction are listed below.

Bioactive Factors to Promote Immune Responses

a. Interleukins: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12IL-15, IL-17, IL-18 etc.b. TNF-αc. IFN-γd. IFN-αe. GM-CSFf. G-CSFg. Ftl-3 ligandh. MIP-3 13 (CCL19)i. CCL21j. M-CSFk. MIFl. CD40Lm. CD3n. ICAMo. Anti CTLA-4 antibodiesp. TGF-βq. CPG rich DNA or oligonucleotidesr. Sugar moieties associated with Bacteria: Lipopolysacharides (LPS) isan examples. Fas ligandt. Trailu. Lymphotactinv. Mannan (M-FP)w. Heat Shock Proteins (apg-2, Hsp70 and Hsp 90 are examples)

Diseases and Antigens—Vaccination Targets

a. Cancer: antigens and their sourcesi. Tumor lysates extracted from biopsiesii. Irradiated tumor cellsiii. Melanoma1. MAGE series of antigens (MAGE-1 is an example)

2. MART-1/melana 3. Tyrosinase

4. ganglioside5. gp100

6. GD-2 7. O-acetylated GD-3 8. GM-2

iv. Breast Cancer

1. MUC-1 2. Sos 1

3. Protein kinase C-binding protein4. Reverse trascriptase protein5. AKAP protein

6. VRK1 7. KIAA1735 8. T7-1, T11-3, T11-9

v. Other General and Specific Cancer Antigens1. Homo Sapiens telomerase ferment (hTRT)

2. Cytokeratin-19 (CYFRA21-1) 3. SQUAMOUS CELL CARCINOMA ANTIGEN 1(SCCA-1), (PROTEIN T4-A) 4. SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2)

5. Ovarian carcinoma antigen CAl25 (1A1-3B) (KIAA0049)6. MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN),(POLYMORPHIC EPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN),(TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG),(PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATEDANTIGEN DF3)7. CTCL tumor antigen se1-18. CTCL tumor antigen se14-39. CTCL tumor antigen se20-410. CTCL tumor antigen se20-911. CTCL tumor antigen se33-112. CTCL tumor antigen se37-213. CTCL tumor antigen se57-114. CTCL tumor antigen se89-115. Prostate-specific membrane antigen16. 5T4 oncofetal trophoblast glycoprotein17. Orf73 Kaposi's sarcoma-associated herpesvirus18. MAGE-C1 (cancer/testis antigen CT7)

19. MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10) 20. MAGE-B2 ANTIGEN (DAM6)21. MAGE-2 ANTIGEN

22. MAGE-4a antigen23. MAGE-4b antigen24. Colon cancer antigen NY-CO-4525. Lung cancer antigen NY-LU-12 variant A26. Cancer associated surface antigen27. Adenocarcinoma antigen ART128. Paraneoplastic associated brain-testis-cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen)29. Neuro-oncological ventral antigen 2 (NOVA2)30. Hepatocellular carcinoma antigen gene 520

31. TUMOR-ASSOCIATED ANTIGEN CO-029

32. Tumor-associated antigen MAGE-X233. Synovial sarcoma, X breakpoint 234. Squamous cell carcinoma antigen recognized by T cell35. Serologically defined colon cancer antigen 136. Serologically defined breast cancer antigen NY-BR-1537. Serologically defined breast cancer antigen NY-BR-1638. Chromogranin A; parathyroid secretory protein 1

39. DUPAN-2 40. CA 19-9 41. CA 72-4 42. CA 195

43. Carcinoembryonic antigen (CEA)b. AIDS (HIV Associated Antigens)i. Gp120ii. SIV229iii. SIVE660iv. SHIV89.6Pv. E92vi. HClvii. OKMSviii. FVIIIRAgix. HLA-DR (Ia) antigensx. OKM1xi. LFA-3c. General Infectious Diseases and Associated Antigensi. Tuberculosis1. Mycobacterium tuberculosis antigen 52. Mycobacterium tuberculosis antigen 85

3. ESAT-6 4. CFP-10 5. Rv3871 6. GLU-S

ii. Malaria

1. CRA 2. RAP-2 3. MSP-2 4. AMA-1

iii. Possible mutant influenza and meningitis strainsd. Neuro Protection—Protect Against Neurological Diseases (e.g.,Alzheimer's, Parkinsons, Prion Disease)1. Classes of self CNS antigens2. human alpha-synuclein (Parkinson's)3. beta amyloid plaques (Alzheimer's)e. Autoimmune Diseases (multiple sclerosis, Rheumatoid arthritis etc)i. Disease linked MHC antigensii. Different classes of Self antigensiii. Insuliniv. Insulin peptide B9-23v. glutamic acidvi. decarboxylase 65 (GAD 65)vii. HSP 60Disease linked T-cell receptor (TCR)

In Situ Regulation of DC Subsets and T Cells Mediates Tumor Regressionin Mice

Prior vaccines have been largely ineffective for patients withestablished cancer, as advanced disease requires potent and sustainedactivation of CD8⁺ cytotoxic T lymphocytes (CTLs) to kill tumor cellsand clear the disease. Subsets of dendritic cells (DCs) specialize inantigen cross-presentation and in the production of cytokines, whichregulate both CTLs and T regulatory (Treg) cells that shut down effectorT cell responses. Coordinated regulation of a DC network, andplasmacytoid DCs (pDCs) and CD8⁺ DCs in particular, enhances hostimmunity in mice. Functionalized biomaterials incorporating variouscombinations of an inflammatory cytokine, immune danger signal, andtumor lysates were used to control the activation and localization ofhost DC populations in situ. The numbers of pDCs and CD8⁺ DCs, and theendogenous production of interleukin-12, all correlated strongly withthe magnitude of protective antitumor immunity and the generation ofpotent CD8⁺ CTLs. Vaccination by this method maintained local andsystemic CTL responses for extended periods while inhibiting FoxP3 Tregactivity during antigen clearance, resulting in complete regression ofdistant and established melanoma tumors. The efficacy of this vaccine asa monotherapy against large invasive tumors is a result of the localactivity of pDCs and CD8⁺ DCs induced by persistent danger and antigensignaling at the vaccine site. These results indicate that a criticalpattern of DC subsets correlates with the evolution of therapeuticantitumor responses. Provision of secondary immunostimulatory site oftumor antigen presentation allows one to manipulate the in situgeneration of a heterogeneous DC network capable of CTL induction, andactivate robust CD8 T cell effector responses to established tumors.

Described herein is the in situ generation of a heterogeneous DC networkcapable of CTL induction to activate robust CD8+ T cell effectorresponses to established tumors, by providing a secondaryimmunostimulatory site of tumor antigen presentation. Inflammation orinfection produces DC populations that are not found in the steady state(K. Shortman, S. H. Naik, Steady-state and inflammatory dendritic-celldevelopment. Nat. Rev. Immunol. 7, 19-30 (2007)), suggesting thatstimuli in tissue microenvironments provoke a response from the networkof DCs. The cytokine granulocyte-macrophage colony-stimulating factor(GM-CSF) is present at increased concentrations during inflammation (J.A Hamilton, GM-CSF in inflammation and autoimmunity. Trends Immunol. 23,403-408 (2002); M. C. Dieu, B. Vanbervliet, A. Vicari, J. M. Bridon, E.Oldham, S. Alt-Yahia, F. Briere, A. Zlotnik, S. Lebecque, C. Caux,Selective recruitment of immature and mature dendritic cells by distinctchemokines expressed in different anatomic sites. J. Exp. Med. 188,373-386 (1988)), which causes the recruitment of both monocytes and DCswhile inducing local monocytes to differentiate into DCs (G. Dranoff, E.Jaffee, A. Lazenby, P. Golumbek, H. Levitsky, K. Brose, V. Jackson, H.Hamada, D. Pardoll, R. C. Mulligan, Vaccination with irradiated tumorcells engineered to secrete murine granulocyte-macrophagecolony-stimulating factor stimulates potent, specific, and long-lastinganti-tumor immunity. Proc. Natl. Acad. Sci. U.S.A. 90, 3539-3543 (1993);B. Pulendran, J. Banchereau, S. Burkeholder, E. Kraus, E. Guinet, C.Chalouni, D. Caron, C. Maliszewski, J. Davoust, J. Fay, K. Palucka,Flt3-ligand and granulocyte colony-stimulating factor mobilize distincthuman dendritic cell subsets in vivo. J. Immunol. 165, 566-572 (2000)).

Implantable synthetic polymer matrices (antigen-loaded acellularbiomaterial device) that spatially and temporally control the in vivopresentation of cytokines, tumor antigens, and danger signals wereutilized herein. GM-CSF is released from these polylactide-co-glycolide(PLG) [a Food and Drug Administration (FDA)-approved biomaterial]matrices into the surrounding tissue to recruit DC precursors and DCs.CpG-rich oligonucleotides are immobilized on the matrices as dangersignals, and antigen (tumor lysates) is released to matrix-resident DCsto program DC development and maturation. These matrices quantitativelyregulate DC activation and trafficking in situ and induce prophylacticimmunity against inoculations of murine B16-F10 melanoma cells (P.Schnorrer, G. M. Behrens, N. S. Wilson, J. L. Pooley, C. M. Smith, D.El-Sukkari, G. Davey, F. Kupresanin, M. Li, E. Maraskovsky, G. T. Belz,F. R. Carbone, K. Shortman, W. R. Heath, J. A. Villadangos, The dominantrole of CD8⁺ dendritic cells in cross-presentation is not dictated byantigen capture. Proc. Natl. Acad. Sci. U.S.A. 103, 10729-10734 (2006)).As described herein, this system administered repeatedly over time tocontrols the recruitment and activation of multiple DC and T cellsubsets and is effective as a therapeutic vaccine against establishedtumors.

The following materials and methods were used to generate the datadescribed herein.

Matrix Fabrication

An 85:15, 120-kD copolymer of _(D,L)-lactide and glycolide (PLG)(Alkermes) was utilized in a gas-foaming process to form porous PLGmatrices (L. D. Harris, B. S. Kim, D. J. Mooney, Open pore biodegradablematrices formed with gas foaming J. Biomed. Mater. Res. 42, 396-402(1998)). In brief, PLG microspheres encapsulating GM-′CSF were firstmade with standard double emulsion (S. Cohen, T. Yoshioka, M. Lucarelli,L. H. Hwang, R. Langer, Controlled delivery systems for proteins basedon poly(lactic/glycolic acid) microspheres. Pharm. Res. 8, 713-720(1991)). PLG micro-spheres were then mixed with 150 mg of the porogen,sucrose (sieved to a particle size between 250 and 425 mm), andcompression molded. The resulting disc was allowed to equilibrate withina high-pressure CO₂ environment, and a rapid reduction in pressurecauses the polymer particles to expand and fuse into an interconnectedstructure. The sucrose was leached from the scaffolds by immersion inwater, yielding scaffolds that were 90% porous. To incorporate tumorlysates into PLG scaffolds, the biopsies of B16-F10 tumors that hadgrown subcutaneously in the backs of C57BL/6J mice (Jackson Laboratory)were digested in collagenase (250 U/ml) (Worthington) and suspended at aconcentration equivalent to 10⁷ cells per milliliter after filtrationthrough 40-μm cell strainers. The tumor cell suspension was subjected tofour cycles of rapid freeze in liquid nitrogen and thaw (37° C.) andthen centrifuged at 400 rpm for 10 min. The supernatant (1 ml)containing tumor lysates was collected, incubated with the PLGmicrospheres, and lyophilized, and the resulting mixture was utilized inthe high-pressure CO₂ process to foam macroporous PLG matricesincorporating tumor lysates. To incorporate CpG-ODNs into PLG scaffolds,CpG-ODN 1826, 5′-tccatgacgttcctgacgtt-3′ (SEQ ID NO: 10) (Invivogen) wascondensed with PEI (M_(n)˜60,000) molecules by dropping ODN 1826solutions into PEI solution while vortexing the mixture (L. D. Harris,B. S. Kim, D. J. Mooney, Open pore biodegradable matrices formed withgas foaming J Biomed. Mater. Res. 42, 396-402 (1998); S. Cohen, T.Yoshioka, M. Lucarelli, L. H. Hwang, R. Langer, Controlled deliverysystems for proteins based on poly(lactic/glycolic acid) microspheres.Pharm. Res. 8, 713-720 (1991); Y. C. Huang, M. Connell, Y. Park, D. J.Mooney, K. G. Rice, Fabrication and in vitro testing of polymericdelivery system for condensed DNA. J. Biomed. Mater. Res. A 67,1384-1392(2003)). The charge ratio between PEI and CpG-ODN (NH₃ ⁺:PO₄)was kept constant at 7 during condensation. PEI-CpG-ODN condensatesolutions were then vortexed with 60 μl of 50% (w/v) sucrose solution,lyophilized, and mixed with dry sucrose to a final weight of 150 mg. Thesucrose containing PEI-CpG-ODN condensate was then mixed with blank,GM-CSF, and/or tumor lysate-loaded PLG microspheres to make PLG cancervaccines.

In Situ Identification of DC Subsets and T Cells

Blank PLG matrices and matrices containing 3000 ng of GM-CSF alone or incombination with either 1, 10, 50, or 100 μg of CpG-ODN (studies werealso performed with tumor lysates copresented with either 3000 ng ofGM-CSF or 100 μg of CpG-ODN alone or in combination) were implanted intosubcutaneous pockets on the back of 7- to 9-week-old male C57BL/6J mice.For histological examination, scaffolds were excised and fixed in Z-fixsolution (Anatech), embedded in paraffin, and stained with hematoxylinand eosin (H&E). To analyze DC recruitment, scaffolds were excised atvarious time points and digested the ingrown tissue into single-cellsuspensions with a collagenase solution (250 U/ml; Worthington) that wasagitated at 37° C. for 45 min. The cell suspensions were then pouredthrough a 40-μm cell strainer to isolate cells from scaffold particles,and the cells were pelleted and washed with cold PBS and counted with aZ2 coulter counter (Beckman Coulter). To assess DC infiltration andactivation, subsets of the total cell population isolated from PLGmatrices were stained with primary antibodies (BD Pharmingen) conjugatedto fluorescent markers to allow for analysis by flow cytometry.Allophycocyanin (APC)-conjugated CD11c (DC marker) and phycoerythrin(PE)-conjugated CD86 (B7, costimulatory molecule) stains were conductedfor DC recruitment analysis, and APC-conjugated CD11c, fluoresceinisothiocyanate (FITC)-conjugated CCR7, and PE-conjugated MHCII stainswere conducted for DC programming analysis. To further delineate thepresence of specific DC subsets, cells were stained with APC-conjugatedCD11c and PE-conjugated PDCA-1 (pDC marker), APC-conjugated CD11c andPE-conjugated CD8 (CD8 DCs), or APC-conjugated CD11c and FITC-conjugatedCD11b (CD11b DCs). To assess T cell infiltration, PE-Cy7-conjugated CD3stains were performed in conjunction with APC-conjugated CD8a (CD8 Tcells), FITC-conjugated CD4 (CD4 T cells), and PE-conjugated FoxP3(Treg) and analyzed with flow cytometry. Cells were gated according topositive FITC, APC, and PE with isotype controls, and the percentage ofcells staining positive for each surface antigen was recorded.

Tumor Growth Assays, Protective Cytokines, and TRP2 Pentamer Analysis

PLG scaffolds with melanoma tumor lysates and various dosages of GM-CSFand/or various quantities of PEI-CpG-ODN condensates were implantedsubcutaneously into the lower left flank of C57BL/6J mice. Forprophylactic vaccinations, animals were challenged 14 days later with asubcutaneous injection of 10⁵ B16-F10 melanoma cells [American TypeCulture Collection (ATCC)] in the back of the neck. Animals weremonitored for the onset of tumor growth (˜1 mm³) and killed for humanereasons when tumors grew to 20 to 25 mm (longest diameter).

To assess PLG vaccine efficacy in the therapeutic setting, C57BL/6J micewere challenged with a subcutaneous injection of 5×10⁵ B16-F10 melanomacells (ATCC) in the back of the neck. At either day 9 or day 13 aftertumor challenge, PLG vaccines loaded with 3000 ng of GM-CSF, 100 μg ofCpG-ODN, and tumor lysates were implanted subcutaneously into the lowerleft flank of C57BL/6J mice. A subset of mice was vaccinated again at 10days after the initial vaccination (days 19 and 23).

To determine in vivo IL-12p70, IFN-α, IFN-γ, and TGF-β concentrations atthe matrix implant site, the adjacent tissue was excised and digestedwith tissue protein extraction reagent (Pierce). After centrifugation,the concentrations of IL-12, IFN-α, IFN-γ, and TGF-β in the supernatantwere then analyzed with enzyme-linked immunosorbent assay (R&D Systems)according to the manufacturer's instructions.

To determine the generation of TRP2-specific CTLs, single-cellsuspensions were prepared from the spleens of mice immunized with PLGvaccines (lysate+3000 ng of GM-CSF+100 μg of CpG) at various timepoints. These cells were initially stained with APC-H-2Kb-TRP2 pentamers(Proimmune) and subsequently stained with PE-conjugated monoclonalantibody to CD8 (BD Pharmingen) before being analyzed by flow cytometry.

The data indicate that an implanted copolymer matrix (antigen-loadedacellular biomaterial device) that incorporates inflammatory cytokines,immune danger signal, and tumor antigens elicits an immune responsenetwork that eradicates established tumors in vivo.

Statistical Analysis

All values in the present study were expressed as mean±SD. Thesignificant differences between the groups were analyzed by a Student'st test and a P value of <0.05 was considered significant.

Local GM-CSF Delivery Promotes Recruitment of CD11 DCs

Macroporous PLG matrices were fabricated for GM-CSF release to recruitDCs and with an interconnected porous structure facilitates cellinfiltration. Matrices were loaded with 0, 3000, and 7000 ng of GM-CSFand implanted into the subcutaneous pockets of C57BL/6J mice.Histological analysis at day 14 after implantation of PLG matricesloaded with 3000 ng of GM-CSF revealed enhanced cellular infiltrationwhen compared to blank controls. Fluorescence-activated cell sorting(FACS) analysis for CD11c DCs showed that GM-CSF delivery recruitedsignificantly more DCs (a factor of ˜8 increase) than blank PLGmatrices. The matrix-resident DCs were almost exclusively CD11b⁺ (87%),in accordance with other studies of GM-CSF effects on DC recruitment invivo (N. Mach, S. Gillessen, S. B. Wilson, C. Sheehan, M. Mihm, G.Dranoff, Differences in dendritic cells stimulated in vivo by tumorsengineered to secrete granulocyte-macrophage colony stimulating factoror Flt3-ligand. Cancer Res. 60, 3239-3246 (2000); E. Daro, B. Pulendran,K. Brasel, M. Teepe, D. Pettit, D. H. Lynch, D. Vremec, L. Robb, K.Shortman, H. J. McKenna, C. R. Maliszewski, E. Maraskovsky, Polyethyleneglycolmodified GM-CSF expands CD11bhighCD11chigh but notCD11blowCD11chigh murine dendritic cells in vivo: A comparative analysiswith Flt3 ligand. J. Immunol. 165, 49-58 (2000)). The total number ofDCs recruited and their expression of the costimulatory molecule CD86increased with GM-CSF delivery in a dose-dependent manner However, thehighest dose (7000 ng) of GM-CSF reduced the number of activated DCs atthe implant site, as indicated by diminished major histocompatibilitycomplex class II (MHCII) and CCR7 expression at day 14 afterimplantation. Because total DC recruitment and activation both peaked at3000 ng of GM-CSF, this dose was utilized to recruit and generate DCs.GM-CSF delivery promoted greater cellular penetration into andassociation with the PLG material, as indicated by histological analysisand measurement of DC numbers, allowing for the subsequent programmingof resident DC precursors and DCs.

In Situ Delivery of CpG-Oligodeoxynucleotide Promotes pDC Recruitmentand IFN Production

The ability of local presentation of danger signals to regulate theratio of distinct DC subtypes was next examined by immobilizingTLR-activating, polyethylenimine (PEI)-condensedCpG-oligodeoxynucleotide (ODN) molecules into the matrices. Condensationof oligonucleotides with the polycationic polymer PEI results inpositively charged particles that bind electrostatically to the anionicPLG matrix. PLG matrices incorporating CpG-ODN alone recruitedCD11c⁺-PDCA-1⁺-pDCs to the PLG matrix. This effect was enhanced withcoadministration of GM-CSF. The dose of CpG-ODN presented in combinationwith 3000 ng of GM-CSF was altered to regulate the numbers of residentpDCs, resulting in 190,000, 520,000, and 1,200,000 cells at doses of 0,10, and 100 μg of CpG-ODN, respectively. Copresentation of CpG-ODN hadlittle effect on the ability of GM-CSF to enhance CD11c⁺-CD11b⁺ cDCs.High doses of CpG-ODN promoted the local production of IFN-α (1010μg/ml) and IFN-γ (˜600 μg/ml) independently of the presence of GM-CSF.These results indicate that controlled GM-CSF and CpG-ODN dangersignaling from synthetic extracellular matrices cooperates to regulateresident pDC and CD11c⁺ CD11b⁺ cDC numbers, along with the production ofprotective cytokines commonly linked to TH1 and CTL immunity.

Tumor Lysate Co-Delivery with CpG-ODN and GM-CSF Stimulates CD8+Generation and IL-12 Production

Experiments were carried out to determine whether co-presenting cancerantigens with CpG-ODNs to matrix-resident DCs would promote further DCdevelopment, activation, and CTL antigen sensitization. In this context,necrotic tumor cells may be particularly immunostimulatory, as theyrelease a variety of endogenous mediators (for example, heat shockproteins and damaged nucleic acids) that trigger innate immunerecognition (C. Fonseca, G. Dranoff, Capitalizing on the immunogenicityof dying tumor cells. Clin. Cancer Res. 14, 1603-1608 (2008)). Thus,freeze-thaw lysates of B16 melanomas were prepared, andantigen-presenting matrices were fabricated by encapsulating theselysates into the PLG material, resulting in localized and sustainedantigen presentation to the infiltrating cell population (O. A. Ali, N.Huebsch, L. Cao, G. Dranoff, D. J. Mooney, Infection-mimicking materialsto program dendritic cells in situ. Nat. Mater. 8, 151-158 (2009)).These antigen-presenting matrices unexpectedly stimulated CD8⁺ DCgeneration in situ). On viral invasion, CD8⁺ CD11c⁺ cDCs are especiallyefficient at cross-presenting exogenous antigen on MHCII molecules (J.D. Farrar, H. Asnagli, K. M. Murphy, T helper subset development: Rolesof instruction, selection, and transcription. J. Clin. Invest. 109,431-435 (2002); D. Skokos, M. C. Nussenzweig, CD8 DCs induceIL-12-independent Th1 differentiation through Delta 4 Notch-like ligandin response to bacterial LPS. J. Exp. Med. 204, 1525-1531 (2007); J. M.den Haan, S. M. Lehar, M. J. Bevan, CD8⁺ but not CD8⁻ dendritic cellscross-prime cytotoxic T cells in vivo. J. Exp. Med. 192, 1685-1696(2000)) and at producing the T_(H)1-promoting cytokine IL-12 (M. Moser,K. M. Murphy, Dendritic cell regulation of TH1-TH2 development. Nat.Immunol. 1, 199-205 (2000); D. Jankovic, M. C. Kullberg, S. Hieny, P.Caspar, C. M. Collazo, A. Sher, In the absence of IL-12, CD4+ T cellresponses to intracellular pathogens fail to default to a Th2 patternand are host protective in an IL-10^(−/−) setting. Immunity 16, 429-439(2002); V. E. Schijns, B. L. Haagmans, C. M. Wierda, B. Kruithof, I. A.Heijnen, G. Alber, M. C. Horzinek, Mice lacking IL-12 develop polarizedTh1 cells during viral infection. J. Immunol. 160, 3958-3964 (1998); J.Magram, J. Sfarra, S. Connaughton, D. Faherty, R. Warrier, D. Carvajal,C. Y. Wu, C. Stewart, U. Sarmiento, M. K. Gately, IL-12-deficient miceare defective but not devoid of type 1 cytokine responses. Ann. N.Y.Acad. Sci. 795, 60-70 (1996)), which are two mechanisms that aid inpriming CTL immunity to viruses and tumors. This activity, however, isnormally associated with lymphoid tissues. Co-presentation of tumorlysates with CpG-ODN led to the presence of 200,000 CD8⁺ DCs, whichincreased to −670,000 (a factor of 9 increase over blank matrices) whenGM-CSF was added to stimulate recruitment. Additionally, tumor lysate incombination with GM-CSF and CpG enhanced the numbers of recruited pDCsat day 10 after implantation by a factor of 2 over matrices withoutlysate and by a factor of 10 over blank controls. No significantdifference in pDC numbers was observed with tumor lysate in combinationwith only GM-CSF or CpG signaling. The CD11c⁺ CD11b⁺ DC population atthe vaccine site depended on GM-CSF alone, as tumor lysate or CpGsignaling alone or in combination had no significant effect on therecruitment and expansion of these DCs.

In situ production of the T cell growth factor IL-12 at matrices thatdeliver both tumor lysate and CpG-ODN to cell populations recruited byGM-CSF was about four times at blank matrices and at least twice at allother matrix formulations. However, tumor lysates in the matrix did notincrease the high concentrations of IFN-α and IFN-γ induced by CpG-ODNalone. These results indicate that the engineered matrices manipulatedboth the number and the function of specific DC subsets, as well as theaccompanying CTL-polarizing activity.

PLG Matrices Co-Delivering GM-CSF, CpG-ODN, and Tumor Lysates StimulatePotent Local and Systemic CD8+ Cytotoxic T Cells

To elucidate the adaptive immune mechanisms induced by PLG vaccines thatdeliver tumor lysate, GM-CSF, and CpG-ODN, the activity of both localand systemic CTLs was examined. Flow cytometric analysis of cellsinfiltrating the vaccine site revealed a significant CD3⁺ CD8⁺ T cellresponse by day 5 (representative sample: ˜1.9×10⁵ cells), which peakedat day 12 when a relatively large proportion of the matrix-residentcells were CTLs (representative sample: 8.5% of cells; ˜8.5×10⁵ cells).Local CD8⁺ T cell numbers dropped sharply by day 16 and were negligibleat day 21 likely because of antigen clearance. PLG vaccines containingtumor lysates, GM-CSF, and CpG-ODN preferentially tuned and promotedCD8⁺ cytotoxic immune responses relative to other matrix formulationsdevoid of CpG. Further, the activation and persistence of systemic CTLresponses was monitored by staining splenocytes withMHCII-tyrosinase-related protein 2 (TRP2) peptide pentamers to identifyCTLs with specificity to TRP2, which is a major antigenic target ofmelanoma vaccines in mice and humans. A significant expansion ofTRP2-specific CTLs was observed in the spleens of vaccinated mice byday5, which continued and peaked between days 7 and 16 before falling atdays 21 to 28, indicating that systemic anti-melanoma responses werebeing generated and sustained for extended periods.

Tumor Protection Induced by PLG Matrices Correlated with DC Subsets andIL-12 Production

This system is capable of generating prophylactic immunity againstpoorly immunogenic B16-F10 melanoma (0. A. Ali, N. Huebsch, L. Cao, G.Dranoff, D. J. Mooney, Infection-mimicking materials to programdendritic cells in situ. Nat. Mater. 8, 151-158 (2009)). The relation ofthis antitumor efficacy to the specific DC networks invoked by variousvaccine formulations was investigated. C57BL/6J mice were vaccinatedwith PLG-based matrices incorporating B16 tumor lysates, GM-CSF, andCpG-ODN in varying combinations and then challenged with live B16-F10melanoma tumor cells at day 14 after vaccination. PLG vaccines with bothB16-F10 tumor lysates and either 1, 10, 50, or 100 mg doses of CpG-ODNdanger signaling allowed 10 to 30% of the vaccinated mice to survive,tumor-free, after an otherwise lethal cell challenge, whereas 100% ofunvaccinated mice were killed by day 23 due to tumor burden. WhenGM-CSF-mediated DC recruitment was combined with lysate and CpG-ODNdelivery, the mice showed significant protection from tumor-inducedlethality. CpG-ODN doses of 10, 50, and 100 μg resulted in 50%, 60%, and90% survival rates, respectively.

The ability of vaccine systems to create a heterogeneous DC populationcorrelated with the marked increase in antitumor efficacy. In comparisonto antigen matrices delivering GM-CSF alone, the antigen-loaded matricesdelivering CpG and GM-CSF together resulted in a higher proportion ofpDCs (˜31% versus 7%) and CD8⁺ cDCs (˜14% versus 5.5%), which correlatedwith a significant enhancement in mouse survival (90% versus 20%),although total DC numbers in situ were statistically similar (3.0±0.6versus 4 2±0 9 million DCs; two-tailed Student's t test, n=5). Survivalrates were proportional to the number of pDCs and CD8⁺ cDCs, but notCD11b⁺ DCs, generated at the PLG vaccine site at day 10. Additionally,the endogenous production of IL-12 was correlated with animal survival,indicating the importance of cross-presentation and T_(H)1-promotingcytokines to vaccine efficacy.

Engineered PLG Matrices Incorporating CpG-ODN Attenuate ImmuneRegulation by FoxP3 Treg Number and Immunosuppressive Cytokines

Although several vaccines designed to program DCs either ex vivo or insitu have achieved significant and long-term prophylactic protection inmouse models of cancer, eradication of invasive and well-establishedtumors has not been achieved without adoptive T cell transfer orsystemic therapies (W. W. Overwijk, M. R. Theoret, S. E. Finkelstein, D.R. Surman, L. A. de Jong, F. A. Vyth-Dreese, T. A. Dellemijn, P. A.Antony, P. J. Spiess, D. C. Palmer, D. M. Heimann, C. A. Klebanoff, Z.Yu, L. N. Hwang, L. Feigenbaum, A. M. Kruisbeek, S. A. Rosenberg, N. P.Restifo, Tumor regression and autoimmunity after reversal of afunctionally tolerant state of self-reactive CD8⁺ T cells. J. Exp. Med.198, 569-580 (2003); Y. Tamura, P. Peng, K. Liu, M. Daou, P. K.Srivastava, Immunotherapy of tumors with autologous tumor-derived heatshock protein preparations. Science 278, 117-120 (1997)). Thislimitation might reflect, at least in part, the ability of DC-basedvaccines to stimulate Treg cells (S. A. Quezada, K. S. Peggs, M. A.Curran, J. P. Allison, CTLA4 blockade and GM-CSF combinationimmunotherapy alters the intratumor balance of effector and regulatory Tcells. J. Clin. Invest. 116, 1935-1945 (2006); M. Jinushi, Y. Nakazaki,M. Dougan, D. R. Carrasco, M. Mihm, G. Dranoff, MFG-E8-mediated uptakeof apoptotic cells by APCs links the pro- and antiinflammatoryactivities of GM-CSF. J. Clin. Invest. 117, 1902-1 913 (2007)) thatattenuate the cytotoxic activity of adaptive immune responses. Thus, theimpact of the engineered matrices on the induction of immunosuppressivepathways was examined Monitoring CD4⁺ T cell responses toantigen-presenting matrices with GM-CSF and CpG revealed peak activityat days 5 and 7, which decreased to negligible concentrations by day 12after implantation. By contrast, matrices containing GM-CSF and tumorlysate led to a significant enhancement of CD4 T cell infiltration atday 12, and these cells likely contribute to regulation of CTLresponses. Incorporation of GM-CSF and tumor lysate into the vaccinematrix led to a factor of 10 increase in TGFβ concentrations and asignificant increase in IL-10 at the vaccine site; these are cytokinescommonly associated with Treg activity and immunosuppression. Further,as observed previously in GM-CSF-based vaccines, GM-CSF cosignaling withtumor antigens resulted in a significant CD3⁺FoxP3⁺ response at thevaccine site when compared to all other matrix formulations, resultingin an almost even ratio of CD8⁺ effectors and FoxP3 Treg cells. CpG-ODNpresentation in concert with both tumor lysate and GM-CSF counteractedthese immunosuppressive mechanisms, as TGFβ and IL-10 concentrations andTreg activity were not enhanced over the control matrices, and CD8 CTLsoutnumbered FoxP3 T cells by a factor of −25 at day 12 afterimplantation. These findings indicate that the vaccine system promotesand extends CTL responses through naïve T cell differentiation inducedby pDCs and CD8⁺ DCs, the corresponding production of type 1 IFNs andIL-12, and inhibition of negative feedback mechanisms.

EXAMPLES Example 1 PLG Devices Loaded with GM-CSF

PLG matrices loaded with 3 μg of GM-CSF were implanted into thesubcutaneous pockets of C57BL/6J mice. The macroporous PLG matrixpresents GM-CSF, danger signals, and cancer antigens in a definedspatiotemporal manner in vivo, and serves as a residence for recruitedDCs as they are programmed. These matrices released approximately 60% oftheir bioactive GM-CSF load within the first 5 days, followed by slowand sustained release of bioactive GM-CSF over the next 10 days (FIG.11A) to effectively recruit resident DCs.

The matrices were made as follows. A 85:15, 120 kD copolymer ofD,L-lactide and glycolide (PLG) (Alkermes, Cambridge, Mass.) wasutilized in a gas-foaming process to form macroporous PLG matrices(Harris, L. D., Kim, B. S., and Mooney, D. J. Open pore biodegradablematrices formed with gas foaming. J. Biomed. Mater, Res, 42, 396-402(1998)). GM-CSF was encapsulated (54% efficiency) into PLG scaffoldsusing a high pressure CO₂ foaming process. PLG microspheresencapsulating GM-CSF were made using standard double emulsion (Cohen S.,Yoshioka T., Lucarelli, M., Hwang L. H., and Langer R. Controlleddelivery systems for proteins based on poly(lactic/glycolic acid)microspheres. Pharm. Res. 8, 713-720 (1991)). To incorporate tumorlysates, biopsies of B16-F10 tumors that had grown subcutaneously in thebacks of C57BL/6J mice (Jackson Laboratory, Bar Harbor Me.), weredigested in collagenase (250 U/ml) (Worthington, Lakewood, N.J.), andsubjected to 4 cycles of rapid freeze in liquid nitrogen and thaw (37°C.) and then centrifuged at 400 rpm for 10 min. The supernatantcontaining tumor lysates was collected and lyophilized with the PLGmicrospheres and the resulting mixture was used to make PLGscaffold-based cancer vaccines. To incorporate CpG-ODNs into PLGscaffolds, CpG-ODN 1826, 5′-tcc atg acg ttc ctg acg tt-3′, (SEQ ID NO:10; Invivogen, San Diego, Calif.) was first condensed withpoly(ethylenimine) (PEI, Mw—25,000 g mol-1, Sigma Aldrich) molecules bydropping ODN-1826 solutions into PEI solution, while vortexing themixture. The charge ratio between PEI and CpG-ODN (NH3+:PO4−) was keptconstant at 7 during condensation. PEI-CpG-ODN condensate solutions werethen vortexed with 60 μl of 50% (wt/vol) sucrose solution, lyophilizedand mixed with dry sucrose to a final weight of 150 mg. The sucrosecontaining condensates was then mixed with blank, GM-CSF and/or tumorlysate loaded PLG microspheres to make PLG cancer vaccines.

Following administration to the animals, histological analysis wascarried out at day 14. The analysis revealed that the total cellularinfiltration into scaffolds was significantly enhanced compared tocontrol (no incorporated GM-CSF) (FIG. 11B). Analysis for DCsspecifically (cells positive for cell surface antigens CD11c and CD86)showed that GM-CSF increased not just the total resident cell number,but also the percentage of cells that were DCs (FIG. 11C). The number ofDCs residing in the material as a result of GM-CSF delivery wasapproximately the same or better than the number of DCs that arecommonly programmed and administered by ex vivo protocols (˜10⁶ cells),and enhanced DC numbers were sustained in the material over time. Theeffects of GM-CSF on in vivo DC recruitment were time and dose-dependent(FIG. 11D).

The dose of GM-CSF delivered from the PLG scaffolds was altered toprovide distinct in vivo concentration profiles in the surroundingtissue, and regulate DC maturation and dispersion of resident DCs (FIG.11E) Implantation of scaffolds with no GM-CSF led to moderate locallevels immediately after implantation that subsequently fell to lowlevels by day 1-2, and then peaked again at day 5, likely due to theinflammatory response to the surgery and implanted PLG. Delivery ofGM-CSF from the PLG scaffolds led to a similar GM-CSF concentrationprofile over time, but at much higher local concentrations. Byapproximately doubling the initial dose of GM-CSF, the system attainedan order of magnitude difference in the peak levels of GM-CSF in vivo,likely due to endogenous GM-CSF production by resident DCs andleukocytes. The secondary peak for GM-CSF was found at day 5 for the3000 ng dose, and at day 7 for the 7000 ng dose (FIG. 11E). Regardlessof whether 3000 or 7000 ng doses of GM-CSF were utilized, the activationstate of DCs peaked when GM-CSF levels began to subside (at days 10 and28, respectively) and enter into the optimal concentration range for DCprogramming

The ability of the pulse of GM-CSF to recruit and subsequently release abatch of activated DCs to home to the lymph nodes was then tested.Fluorescein isocyanate (FITC) was incorporated into and painted onto PLGscaffolds, as DCs recruited to the scaffold ingest this label. The labelcan be later used to identify these cells following their trafficking tothe inguinal lymph nodes. At day 2, the 3000 ng dose of GM-CSF led to aninhibition of lymph node homing, likely due to the high initial levelsof GM-CSF that entrap DCs at the scaffold site (FIG. 11F). However, asGM-CSF levels subsided, a batch of the recruited, FITC-positive DCs werereleased from the matrices, resulting in a superior and a sustained DCpresence in the lymph nodes.

As temporally controlling the local GM-CSF concentration in turncontrols recruitment, and dispersement of a batch of DCs, the utility ofthese cells as a cancer vaccine was evaluated by immobilizing melanomatumor lysates into the matrices to load resident DCs with tumorantigens. These PLG cancer vaccines were implanted into C57BL/6J mice,and 14 days later these mice were injected with highly aggressive andmetastatic B16-F10 melanoma cells. All mice implanted solely with blankPLG scaffolds had appreciable tumors within 18 days and had to beeuthanized by day 23, due to the aggressiveness of these cells. Deliveryof antigen alone from the PLG scaffolds slightly improved the fate ofthe mice, as some mice in this group survived until day 40.Surprisingly, co-delivery of GM-CSF with antigen dramatically decreasedtumor formation, and the optimal GM-CSF dose delayed tumor formation byapproximately 40 days in 50% of the animals, and cured 23% of animals.Moreover, localized tumor antigen presentation in combination withoptimal GM-CSF exposure (400 ng) increased the average time before tumorformation by 3-fold as compared to antigen alone, and by nearly 2-foldover non-optimal GM-CSF exposure.

Analysis of T-cell infiltration into tumor tissue byimmunohistochemistry was next performed to determine if programmed DCswere capable of inducing T-cell activation and homing to tumors.Vaccination with antigen alone resulted in CD4(+) T-cell infiltrates.Notably, recruiting and programming a batch of DCs in situ withappropriate GM-CSF presentation resulted in a 2-fold increase in CD8(+)cytotoxic T-cell numbers over blank controls. The vaccine's efficacy wasattenuated in CD8 and CD4 T-cell knock-out mice, attesting to thespecific role of CD4 and CD8 T-cells in the immune protection.

A continuous process of in situ DC programming is achieved by presentingadditional cues that released the DCs from GM-CSF inhibition once theyreside in the matrices. In particular, the presentation of syntheticCpG-ODN with exogenous GM-CSF provides a mimic of bacterial infections,in which cells recruited by inflammatory cytokines are stimulated bylocal toll-like receptor activating “danger signals”, such as CpG-ODNpresent in bacteria. CpG-ODN was immobilized to the PLG matrices byfirst condensing nucleotides with polyethylenimine (PEI) to formcationic nanoparticles. Following foaming of a combination of CpG-ODNand PLG particles, the CpG-ODN was largely retained in the matrices(>80% over 25 days) due to electrostatic interactions with the anionicPLG material. The CpG-ODN immobilization allows for host DCs, recruitedby GM-CSF, to uptake these nucleotides locally as they reside in thematrices. Surprisingly, this approach resulted in approximately 2.5 and4.5 fold increases in the numbers of activated DCs (positive for MHCIIand CCR7) in the scaffolds, respectively, over GM-CSF or CpG-ODNdelivery alone. CpG-ODN presentation enhanced DC activation in thepresence of inhibitory GM-CSF levels (>40 ng/ml) in situ, indicating amore continuous process of DC recruitment and activation. Thisinfection-mimicking system reliably generated activated DCs′. Themagnitude of the immune response with this infection-mimic was confirmedgrossly, as the lymph nodes of these animals were markedly enlarged.Most importantly, a 6-fold increase in the number of DCs that were firstrecruited to the matrices and subsequently dispersed to the lymph nodeswas achieved with this system.

The ability of continuous DC recruitment, and programming to generate animmune response was next tested in the melanoma model. The vaccineprovided significant protection, and the level of protection correlatedwith the CpG dose. Animal survival increased from 23% to 50% and finally90% at CpG doses of 0 μg, 10 μg and 100 μg, respectively. This materialinfection-mimic induced equivalent or better immune protection than thatobtained with existing cell-based therapy. Materials presenting CpG-ODNwith lysates alone had only a 20% survival, indicating the benefit ofrecruiting DCs with GM-CSF. The benefit of providing a residence forrecruited DCs while they are programmed was demonstrated by the failureof vaccine formulations consisting of bolus injections of tumor lysates,CpG-ODN, with and without 3000 ng of GM-CSF. Moreover, injecting GM-CSFloaded PLG microspheres to provide sustained GM-CSF delivery withoutproviding a residence for recruited cells, with bolus CpG-ODN and tumorlysate delivery resulted in little immune protection and animals did notsurvive over 35 days.

To further examine the mechanism of immune protection with this materialsystem, the subsets of DCs and the endogenous production of cytokines bythese cells in materials presenting GM-CSF and CpG-ODN alone or togetherwere analyzed, along with the specificity of the immune response. Thedelivery of GM-CSF alone enhanced the recruitment of CD11c(+)CD11b(+)myeloid DCs, whereas CpG-ODN delivery alone had little effect on theoverall numbers of this subset. CpG-ODN delivery did, though, increasethe number of plasmacytoid DCs at the site, which have been described topredominantly secrete Thelper(Th)-1 cytokines, especially typelinterferons and interleukin(IL)-12 that can promote CD8(+), cytotoxic Tcell immunity in response to CpG-ODN presentation with antigen.Accordingly, CpG signaling not only upregulated the expression ofactivation markers on resident DCs, but also induced IFN-γ and IL-12production at the vaccine site, as expected from the increased presenceof plasmacytoid DCs. Moreover, analysis of T cell infiltrates intotumors that formed in the subset of animals that were not completelyprotected (infection mimics; 10 μg CpG-ODN dose) revealed that, even inthese animals, DC programming with CpG-ODN resulted in an almost 3-foldincrease in CD8(+) T-cell infiltration over controls. Further,tyrosinase-related protein (TRP)-2 is a main antigenic target of theimmune response elicited by melanoma vaccines in both mice (includingB16 whole cell vaccines) and humans, and staining cells isolated fromspleens with MHC class I/TRP2 peptide pentamers revealed a dramaticexpansion of TRP2-specific CD8 T cells in vaccinated mice. Theseantigen-specific T cells are involved in the killing of tumor cells, andfacilitated immune protection after vaccination. Additionally, 33% ofsurviving mice developed patches of skin and hair depigmentationstarting at the sites of tumor inoculation (back of neck).Depigmentation, which likely involves T cell responses to melanocyteantigens, has been correlated to improved clinical responses in humanmelanoma patients, and, in these studies, was only observed in micetreated with infection mimics.

These results indicate that mimicking aspects of infection withpolymeric material systems dramatically impacts tumor progression byeffectively recruiting, activating and homing DCs to lymph nodes. Thefirst approach utilized a pulse of GM-CSF alone to recruit DCs to thetumor-antigen presenting material. The DCs subsequently resided withinthe material and were trapped until GM-CSF levels fell and cells couldbecome activated and disperse. The specific concentration and durationof GM-CSF are critical to its effects. A continuous process wassubsequently developed to shuttle DCs through an infectious-likemicroenvironment via recruitment with GM-CSF, followed by activation ofresident DCs via CpG-ODN presentation, and subsequent release. Thepresentation of PEI condensed CpG-ODN from the material dramaticallyincreased not only the numbers of activated, host DCs residing in thematerial, but also the percentage and total numbers of programmed DCsthat emigrated to the lymph nodes. Further, CpG-ODN signaling selectedfor specific DC subsets and DC functions associated with protectiveimmune responses.

The system's quantitative control over DC trafficking and activationtranslated to a regulation over the efficacy of the cancer vaccine. Asthe numbers of DCs that were programmed and dispersed to the lymph nodesincreased, the survival increased from 0 to 25 and finally 90%. T-cellsmediated immune protection, as a clear relation between the numbers of Tcells in the tumors that did form and vaccine efficacy was found, andinfection mimics induced the generation of melanoma-antigen specific Tcells. The matrix structure was necessary to produce long-lastingimmunity, as vaccines delivered in bolus form and sustained releasewithout provision of a cell residence failed to produce significantprotective immunity. Although reports concluded that either celltransplantation or multiple systemic injections are necessary to promoteprotective immunity in clinically relevant tumor models, the dataindicate that devices comprising functional polymeric residencematerials provide significant and specific immune protection that isequal to or superior to previous systems, even with single applicationat vastly reduced total drug doses (e.g., 3 μg in the scaffold systemvs. 100's μg total dose in repeated, systemic injections).

These data have significant clinical relevance, as the material systemprogrammed DCs in situ, and not only bypassed the complication and costof ex vivo cell manipulation and transplantation, but also providedtight control over the number of DCs recruited, activated and dispersedto the lymph nodes. Patients are treated with and the devices provide analternative to current cancer vaccines, or are used in concert withthose and other approaches.

The system is applicable to other situations in which one desires topromote a destructive immune response (e.g., eradicate infectiousdiseases) or to promote tolerance (e.g., subvert autoimmune disease).The use of polymers as a temporary residence for in situ cellprogramming is a powerful alternative to current cell therapies thatdepend on ex vivo cell manipulation (e.g., stem cell therapies).

Example 2 Condensation of Synthetic CpG-ODN Molecules Increases CellularUptake

Synthetic CpG-ODN molecules were condensed with PEI, which resulted inpositively charged, small PEI-CpG-ODN condensates that facilitatescellular internalization via promoting association with the cellmembrane and enhancing transmembrane transport (FIGS. 2A-C). ODNCondensation at charge ratios of 7 and 15, between the amine groups ofPEI and the phosphate groups of ODNs, resulted in optimal particle sizesand positive charge (FIGS. 2B and C), but a charge ratio of 7 wasutilized in experiments due to PEI toxicity at high doses.

PEI condensation of CpG-ODN dramatically enhanced nucleotide uptake intoDCs in vitro (FIGS. 3A-C). Quantification of CpG-ODN uptake into DCsrevealed orders of magnitude differences (up to −100-fold) between ODNcondensates and naked ODN, which were maintained for extended timeperiods (>80 hrs) in vitro (FIG. 3C). The complexes subsequentlydecondense (FIG. 3D) allowing for CpG-ODN localization to itsintercellular receptor, TLR-9, which has been previously demonstrated tobe present in endosomes.

Example 3 CpG-ODN Induced DC Activation and DC Mobilization

Because effective CpG stimulation of DCs requires intercellularlocalization, the effects of PEI-condensation were evaluated on DCactivation. DCs stimulated with PEI-CpG-ODN in vitro exhibited enhancedlevels of CD86, MHCII and CCR7 expression, in comparison to thosestimulated with naked CpG-ODN, which correlated strongly with DC uptakeof condensates (FIGS. 4A and B). DCs exhibited an activated morphology,upon cellular uptake of PEI-CpG-ODN including the development of fineneedle-like dendrites and large membrane expansion, which allows matureDCs to “wrap-up” T-cells promoting strong cell-cell interactions. Theactivation states of PEI-CpG-ODN stimulated DCs mirrored or surpassedthat of positive controls stimulated with TNF-α and LPS (FIG. 3C) andPEI-CpG-ODN condensates promoted a 3-fold increase in DC migrationtoward CCL19 in vitro, over unstimulated DCs (FIG. 4D).

PEI-CpG-ODN condensates also released DCs from GM-CSF inhibition, assignificant DC activation was noted in cells exposed to both condensedoligonucleotides and high levels of GM-CSF (FIG. 5A). Additionally,PEI-CpG-ODN stimulation also promoted DC migration away from high GM-CSFsources (500 ng/ml) toward CCL19 (FIG. 5B).

A PLG system was developed that effectively immobilized and presentedPEI-CpG-ODN condensates (FIG. 6A) to resident DCs to stimulate DCactivation and mobilization. Local PEI-CpG-ODN presentation promoted DCmobilization in vitro (FIGS. 6A-C). Interestingly, there is an optimaldose range, 5-50 μg, of PEI-CpG-ODN that enhanced DC emigration from PLGmatrices toward CCL19, but high doses (500 μg) had no effect on DCmigration (FIGS. 6B and C). A 25 μg of PEI-CpG-ODN also counteracted thesuppressive effects that high GM-CSF levels had on DC migration, in thismodel (FIG. 6C). These results indicate that appropriate CpG-ODNpresentation provides an avenue to continuously program and dispersehost DCs that are recruited and otherwise trapped by high levels ofGM-CSF in situ.

Example 4 Infection-Mimics Continuously Program and Disperse DCs In Vivo

An infection-mimicking system to continuously recruit and program DCswas created by simultaneous release of GM-CSF to attract host DCs to PLGmatrices, while the PEI-CpG-ODN condensates were largely retained in thematrix (>80% over 25 days) (FIGS. 6A-C), likely via electrostaticinteractions as has been shown for plasmid DNA, allowing for recruitedDCs to uptake the complexes locally. Strikingly, when optimized, thisapproach resulted in approximately 2.5 and 4.5 fold increases in thenumbers of MHCII and CCR7 expressing DCs resident in the matrices insitu, respectively (over GM-CSF or CpG-ODN delivery alone) (FIGS. 7A andB). Interestingly, high doses of PEI-CpG-ODN (>50 μg) resulted inrelatively low MHCII expression and enhanced CCR7 expression, indicatingdifferential regulation of DC function in comparison to low doses (FIG.7A). Optimum CpG-ODN signaling (˜10-25 μg) enhanced DC activation in thepresence of inhibitory GM-CSF levels (>40 ng/ml) in situ, and thisinfection-mimicking system generated the numbers of activated DCs (>10⁶)(FIGS. 7A and B) commonly administered in ex vivo protocols.

Most importantly, a 6-fold increase in the number of DCs that were firstrecruited to the matrices and subsequently dispersed to the lymph nodeswas achieved with this system (FIG. 8 A). The magnitude of the immuneresponse with infection-mimics could even be appreciated grossly, as thelymph nodes of these animals were markedly enlarged (FIGS. 8B and C). Ascharacterized by infectious responses, these swollen lymph nodescontained greater numbers of immune cells including DCs (FIGS. 8C andD).

Example 5 Infection-Mimicking Microenvironment Confers Potent Anti-TumorImmunity

The ability of continuous DC recruitment, and programming to generate animmune response was next tested in the melanoma model. This vaccineprovided significant protection, as 50% of the animals did not formtumors over an 80 day time frame (FIG. 9), and this result wasremarkably similar to that obtained with a widely investigatedcell-based therapy (FIG. 9). Animals receiving lys+CpG were 37.5% tumorfree 140 days after treatment and achieved protective immunity.

Furthermore, analysis of T-cell infiltrates into tissue of tumors thatformed in the subset of animals that were not completely protectedrevealed that, even in these animals, DC programming with CpG-ODNresulted in an almost 3-fold increase in CD8(+) T-cell infiltration overcontrols (FIGS. 10A-B). Thus, all animals receiving the Lys-GM-CpGtreatment demonstrated a therapeutic benefit.

Example 6 Tumor Protection is Regulated by CpG-ODN Presentation andPlasmacytoid DC (pDC) Enrichment

Hematopoetic precursor cells of both the myeloid and lymphoid lineagehave the capacity to differentiate into two main categories of DCs,plasmacytoid DCs (pDCs) and conventional DCs (cDCs), each of which areequipped with specific defense mechanisms capable of propagatingspecific responses to invading pathogens. This plasticity likely allowsfor the recruitment and generation of the DC subset(s) most proficientat eliciting the desired immune response. cDCs include CD11c⁺ CD11b⁺ andCD11c⁺ CD8α⁺ cells exhibiting classical DC morphology with the long,protruding dendrites that make them especially adept at antigenprocessing and antigen presentation to T cells. pDCs are roundnon-dendritic cell capable of producing large amounts of type-1interferons in response to ‘danger signals’, such as unmethylated CpGdinucleotide sequences in bacterial or viral DNA.

pDC derived type 1 interferons (IFN) link innate and adaptive immunityto viral infection by triggering antigen cross presentation to CD8+ Tcells and interleukin production (e.g. IL-12) by cDCs that facilitatethe clonal expansion of cytotoxic T cells. Type 1 IFNs also act todirectly induce naïve T cell differentiation to T helper 1 cells. Inaddition to producing potent IFNs, pDCs stimulated by inflammatorystimuli and microbial infection differentiate into a dendritic formcapable of processing and presenting antigen to prime T cell responses.pDCs and cDCs cooperate to perform specialized functions that initiatedistinct cellular and molecular events leading to protective immunity.

Many cell-based vaccines for cancer fail to incorporate the differentcomponents of the DC network. Cancer vaccines are frequently developedusing easily accessible, patient-derived blood monocytes that aretransformed into DCs ex vivo using cytokine mixtures and pulsed withtumor antigens to promote antigen presentation. These antigen-loaded DCsare then injected back into cancer patients with the goal of inducinganti-tumor immune responses mediated primarily by Th1 cells and CTLs.While initial trials utilizing ex vivo DC vaccines in advanced cancerpatients have resulted in antigen-specific T-cell expansion and theproduction of protective cytokines, many vaccines have failed to showsurvival advantage over traditional treatments (e.g., chemotherapy) andhave failed to gain FDA approval. These cell-based vaccines provide nocontrol over the in vivo function of the transplanted DCs and onlyincorporates one DC type into the vaccine, which may not be the mostpotent. Therefore, the rate-limiting step is likely the inability tofully recapitulate ex vivo the development of immunocompetent DCs, inparticular the processes of DC activation and specialization during thegeneration of immune responses. The devices and methods described hereinovercome the shortcomings of such earlier approaches, and therefore,have several advantages over earlier systems.

The devices comprise an implantable, synthetic extra-cellular matrix(ECM) that controls the in situ recruitment and generation of aheterogeneous DC network to produce protective immune responses totumors. GM-CSF was incorporated into polylactide-co-glycolide (an FDAapproved biomaterial) matrices to recruit DC precursors and DCs, as thecytokine is released from the material into the surrounding tissue.These macroporous matrices present immobilized tumor antigens andCpG-rich oligonucleotides as danger signals, capable of programming DCdevelopment and maturation as cells reside within the material. Thedistribution of the DC subsets generated at the vaccine site isregulated by modifying cancer-antigen presentation by the material andthe dosages of danger signals, which significantly affected themagnitude of the protective immune response to tumors when tested in anart recognized B16-F10 tumor model.

Matrices were made to release a pulse of GM-CSF to recruit DCs, and wereloaded with 0, 3000, and 7000 ng of GM-CSF, and implanted into thesubcutaneous pockets of C57BL/6J mice. A GM-CSF gradient formed in thesurrounding tissue, which peaked at 12 hours post-implantation as theGM-CSF concentration reached 100 μg/ml and 30 μg/ml (>30 fold differenceover no incorporated GM-CSF) at distances of 1-3 mm and 3-5 mm,respectively, from the implant site. Elevated GM-CSF levels weremaintained for extended periods (approximately 10 days) while the factorwas released from the PLG to the neighboring tissue. Histologicalanalysis at day 14 post-implantation of PLG matrices loaded with 3000 ngof GM-CSF revealed enhanced cellular infiltration over blank controls,and FACS analysis for the CD11c(+) DC population showed that GM-CSFdelivery recruited significantly more DCs (˜8 fold increase) than blankcontrols. The total number of DCs recruited and their expression of theco-stimulatory molecule CD86 increased with GM-CSF delivery in a dosedependent manner

PLG matrices were then modified to immobilize TLR-activating,PEI-condensed CpG-ODN molecules and present them as danger signals to DCpopulations recruited by GM-CSF. Provision of condensed CpG-ODNsignaling with GM-CSF dramatically enhanced cellular infiltration intoPLG matrices, as revealed by histological analysis at Day 10post-implantation Importantly, CpG-ODN presentation from PLG matricesregulated the local presence of specific DC subsets and the resultingproduction of protective cytokines. Stimulation of the DC infiltraterecruited by GM-CSF with CpG-ODN enriched the PLG matrix withCD11c(+)PDCA-1(+) plasmacytoid DCs (pDCs), a DC subset exhibitingenhanced type 1 IFN production that are associated with t-helper 1 (Th1)immunity.

CpG-ODN leads to preferential recruitment and expansion of pDCs to thetumor site. The dose of CpG-ODN is controlled to regulate the numbers ofresident pDCs, which increased from 190,000, to 520,000, to 1,100,000cells at doses of 0, 10 and 100 μg of CpG-ODN, respectively. GM-CSFdelivery alone significantly enhanced the numbers of CD11c(+)CD11b(+)cDCs recruited to the matrices, but co-presentation of CpG-ODN hadlittle effect on either mDC populations or Cd11c(+)CD8(+) DCs. Highdoses of CpG-ODN promoted the local production of IFN-α (˜1010 μg/ml),IFN-γ (˜600 μg/ml) and, to a lesser degree, IL-12 (150 μg/ml) at theimplant site, which correlated with the increased pDC numbers at thiscondition. The recruitment of DCs by GM-CSF was required for CpG-ODNsignaling to have a significant effect, in terms of expansion of pDCpopulations and production of Th1 cytokines. These results indicate thatcontrolled GM-CSF and CpG-ODN danger signaling from syntheticextra-cellular matrices can effectively regulate resident pDC andCD11c(+)CD11b(+) cDC numbers along with the production of Th1 cytokines.

Studies were carried out to determine whether co-presenting cancerantigens with CpG-ODNs to matrix-resident DCs would promote further DCdevelopment, activation and antigen sensitization, leading to protectivetumor immunity and cytotoxic T cell responses. Antigen-presentingmatrices were fabricated by encapsulating B16-F10 melanoma tumor lysatesinto the PLG matrices. Controlled antigen presentation in combinationwith GM-CSF and CpG signaling enhanced the numbers of resident pDCs atDay 10 post-implantation by 2-fold over matrices without antigen, and by10-fold over blank controls (FIG. 12A). No significant difference in pDCnumbers was observed with antigen presentation in combination withGM-CSF or CpG signaling alone, indicating the benefit of bothGM-CSF-mediated recruitment and CpG-ODN activation of matrix-residentDCs. The CD11c(+)CD11b(+) DC population at the vaccine site depended onGM-CSF delivery alone (FIG. 12B), as antigen or CpG signaling alone orin combination had no significant effect on the recruitment andexpansion of these cDCs (FIG. 12B). Antigen and CpG-ODN presentingmatrices led to the presence of 200,000 CD11c(+)CD8(+) cDCs, whichincreased to approximately 670,000 (9-fold increase over blank matrices)with GM-CSF-mediated recruitment (FIG. 12C). Analysis of the endogenousproduction of IFNs and IL-12 revealed that antigen stimulation incombination with GM-CSF promoted endogenous IFN-α and IFN-γ productionthat was similar to CpG-ODN induction (FIGS. 12D-E). Additionally, thein situ production of the T-cell growth factor, IL-12, at matricespresenting both antigen and CpG-ODN to cell populations recruited byGM-CSF was approximately 4-fold higher than blank matrices at least2-fold higher all other matrix formulations (FIG. 12F). Remarkably, asignificant percentage (10.3%) of the total cells at the site of antigenpresenting matrices were CD8(+) (cDC subset and cytotoxic T-cells) (FIG.12G), which was in correlation with both the number of CD11c(+)CD8(+)cDCs and the concentration of IL-12 (FIGS. 12C, F,G). These resultsindicate that immune responses sensitive to cancer antigen presentationwere generated by manipulating both the number and function of specificDC subsets in situ, including CD8(+)DCs, which was accompanied by CD8+ Tcell activity.

C57BL/6J mice were vaccinated using melanoma antigens (e.g., B16-F10tumor lysates) presented from PLG-based vaccines that differentiallyregulated the generation and function of specific DC subsets in situ(varying GM-CSF and CPG-ODN combinations), and challenged with B16-F10melanoma tumor cells at D14 post-vaccination. PLG vaccines presentingboth B16-F10 tumor lysates and either 1, 10, 50 or 100 μg doses ofCpG-ODN danger signaling led to approximately 10-30% of the vaccinatedmice surviving, tumor-free (FIG. 13A), after an otherwise lethal dosewhile 100% of unvaccinated mice were euthanized by day 23 due to tumorburden. Surprisingly, GM-CSF mediated DC recruitment combined withantigen and CpG-ODN presentation generated significant tumor protection.CpG-ODN doses of 10, 50, and 100 μg resulted in 50, 60 and 90% survivalrates (FIG. 13B). Survival rates correlated strongly with the number ofpDCs generated at the PLG vaccine site at day 10, but did not correlatewith the total CD11c(+)CD11b(+) DC numbers recruited. Additionally, highsurvival rates (60% and 90%) were attained with PLG systems thatgenerated relatively high numbers of CD11c(+)CD8(+) DCs (˜2×10⁵ cells)(FIG. 13E) and increased IFN-α, IFN-γ, and IL-12 production in situ.

The ability of vaccine systems to recruit a heterogeneous DC networkalso had a profound effect on vaccine efficacy, as the DC populationgenerated by CpG and GM-CSF loaded scaffolds compared to GM-CSF loadedscaffolds resulted in a higher proportion of pDCs (˜38% vs. 7%) andCD8+cDCs (˜9.4% vs. 5.5%) (FIG. 13F), leading to a significantenhancement in mouse survival (90% vs. 20%), even though total DCnumbers in situ, were statistically similar (3.05±0.55 vs. 2.67±0.64million DCs). Moreover, tyrosinase-related protein (TRP)-2 is a mainantigenic target of the immune response elicited by melanoma vaccines inboth mice (including B16 whole cell vaccines) and humans, and stainingsplenocytes with MHC class I/TRP2 peptide pentamers revealed asignificant expansion of TRP2-specific CD8 T cells in mice vaccinatedwith GM-CSF, antigen and 100 μg of CpG-ODN (0.55% splenocytes,1.80×10⁵+0.6×10⁴ cells) in comparison to matrices presenting lower CpGdoses, either 0 or 50 μg (0.2% and 0.3% splenocytes). The developmentand expansion of these antigen-specific T cells were induced by thepromotion of pDC activation and their corresponding production of type 1IFNs. These cytotoxic T cells were in turn involved in the killing oftumor cells, which facilitated immune protection after vaccination.These results indicate that devices (PLG matrices) described hereinprecisely regulate the in situ recruitment and expansion of specializedDC subsets. This preferential recruitment and expansion of pDCsdramatically improves immune responses to cancer antigens, reduces tumorprogression, and improves survival of cancer patients compared toprevious vaccine approaches.

FIGS. 14A-B show survival of mice vaccinated with PLG vaccines versuscontrols in a therapeutic model. Mice were innoculated with 5×10⁵ tumorcells and tumors were allowed to grow for 7 days in mice until palpable(1-3 mm³) Mice were vaccinated (at Day 7) with PLG scaffolds containing3 μg GM-CSF, tumor lysates and 100 μg CpG-ODN. Survival data wasobtained using mice (n=10) with established tumors (7 days after tumorinoculation). PLG vaccines containing GM-CSF, lysates and CpG-ODN wereusing for the vaccination. [02] The macroporous, synthetic ECMsdescribed herein provided control over the presentation of inflammatoryand infectious signaling agents creating microenvironments capable ofgenerating distinct DC networks in situ. The total cell number andheterogeneity of these DC networks correlated with the magnitude ofimmune responses to cancer antigens in B16 melanoma models. GM-CSF wasreleased quickly from PLG-based ECMs to recruit and house host DCprecursors and DCs in its macroporous structure. CpG-ODNs were thenimmobilized within the GM-CSF-secreting matrices to direct pDCdevelopment in situ, and, indeed, the CpG signaling not only enhancedCD11c(+)PDCA-1(+) pDC numbers at the implant site, but also enriched thesite with pDCs in a dose dependent manner. When tumor antigen wasincorporated into PLG matrices, enhancement of activity and enrichmentof CD11c+CD8+cDCs at the vaccine site was observed. The provision ofcancer antigens resulted in an enhancement of the total CD8+ cellpopulation, indicating that Cd8+ DCs and Cd8+ T cells responded in situto the antigen-presenting material and that the immune response hadcytotoxic components. Cytokine analysis at the vaccine implant siteindicated that DC subsets act in a cooperative fashion to generate aneffective immune response. pDC numbers correlated strongly with thepresence of type-1 IFNs, which aided the activation of and antigencross-presentation by CD11c(+)CD11b(+) cDCs to enhance CTL priming bythese cells. Additionally, pDCs and CD8+cDC numbers correlated withIL-12 production, which promotes antigen expression andcross-presentation by matrix resident DCs and the development and growthof CTLs.

Tumor growth and T-cell analysis indicated that as the heterogeneity ofthe DC network increased in situ, so did vaccine efficacy. Althoughtotal DC numbers remained statistically similar with GM-CSF signaling,provision of CpG-ODN danger signaling increased pDC numbers in a dosedependent manner, which strongly correlated to animal survival after aB16-F10 tumor challenge. CpG-ODN doses of 10, 50 and 100 μg (in GM-CSFsecreting matrices) along with melanoma antigen presentation from PLGvaccines resulted in 45%, 60% and 90% survival in mice. Removal ofGM-CSF signaling from PLG vaccines sharply reduced the total numbers ofDCs generated in situ, which resulted in survival dropping to 10%,whereas removal of CpG-ODN signaling reduce pDC numbers in situ, as amajority of the DCs (87.4%) were CD11b+ CDCs. The minimum number of DCsrequired to induce protective immunity was determined for each DCsubset, as approximately 600,000 pDCs and 200,000 CD8+cDCs (30% of totalDCs) were required to cooperate with approximately 2,000,0000 CD11b+cDCsto achieve greater than 50% survival after tumor challenge.

The results are clinically significant as the devices and methodsdemonstrated the ability to quantitatively target and employ DC subsetsin vivo for the generation of immunity, resulting in distinct andprotective immune responses.

Example 7 Presentation of TLR Agonists in Structural Polymeric DevicesMaterials & Methods Mice

C57BL/6 mice and Batf3−/− knockout mice (6-8-week-old female; JacksonLaboratories) were used in the studies described in Example 7.

Matrix Fabrication

A 85:15, 120 kD copolymer of D,L-lactide and glycolide (PLG) (Alkermes,Cambridge, Mass.) was utilized in a gas-foaming process to form porousPLG matrices (Harris et al., 1998 J. Biomed. Mater. Res., 42: 396-402).PLG microspheres encapsulating GM-CSF were first made using standarddouble emulsion (Cohen et al., 1991 Pharm. Res., 8: 713-720). The doubleemulsion process was also utilized to fabricate PLG microspherescontaining MPLA (Avanti Polar Lipids, Alabaster, Ala.) as an adjuvant.PLG microspheres were then mixed with 150 mg of the porogen, sucrose(sieved to a particle size between 250 μm and 425 μm), and compressionmolded, thereby yielding a disc device with open, interconnected poresthat are generally of the size range of the porogen. The resulting discwas allowed to equilibrate within a high-pressure CO₂ environment, and arapid reduction in pressure causes the polymer particles to expand andfuse into an interconnected structure (Harris et al., 1998 J. Biomed.Mater. Res., 42: 396-402). The sucrose was leached from the scaffolds byimmersion in water yielding scaffolds that were 90% porous.

To incorporate tumor lysates into PLG scaffolds, biopsies of B16-F10tumors that had grown subcutaneously in the backs of C57BL/6J mice(Jackson Laboratory, Bar Harbor Me.), were digested in collagenase (250U/ml) (Worthington, Lakewood, N.J.) and suspended at a concentrationequivalent to 10⁷ cells per ml after filtration through 40 μm cellstrainers. The tumor cell suspension was subjected to 4 cycles of rapidfreeze in liquid nitrogen and thaw (37° C.) and then centrifuged at 400rpm for 10 min. The supernatant (1 ml) containing tumor lysates wascollected, incubated with the PLG microspheres and lyophilized and theresulting mixture was used to make PLG scaffold-based cancer vaccines.

To incorporate CpG-ODNs and P(I:C) into PLG scaffolds, CpG-ODN 1826,5′-tcc atg acg ttc ctg acg tt-3′, (SEQ ID NO: 10; Invivogen, San Diego,Calif.) or P(I:C) (high molecular weight; Invivogen, Sand Diego, Calif.)was first condensed with poly(ethylenimine) (PEI, Mn˜60,000, SigmaAldrich) molecules by dropping ODN-1826 solutions into a PEI solution,while vortexing the mixture (Huang, et al., 2003 J. Biomed. Mater. Res.,A 67: 1384-1392). The charge ratio between PEI and CpG-ODN (NH3+:PO4−)was kept constant at 7 during condensation. The charge ratio between PEIand P(I:C) (NH3+:PO4−) was kept constant at 3 during condensation. Thecondensate solutions were then vortexed with 60 μl of 50% (wt/vol)sucrose solution, lyophilized and mixed with dry sucrose to a finalweight of 150 mg. The sucrose containing PEI-CpG-ODN condensate was thenmixed with blank, GM-CSF and/or tumor lysate loaded PLG microspheres tomake PLG cancer vaccines.

In Situ Identification of DCs and T Cells

GM-CSF loaded PLG matrices and matrices containing GM-CSF in combinationwith either 100 μg of CpG-ODN, MPLA, or P(I:C) were implanted intosubcutaneous pockets on the back of 7-9 week old male C57BL/6J mice. Toanalyze DC recruitment, scaffolds were excised at various time-pointsand the ingrown tissue was digested into single cell suspensions using acollagenase solution (Worthingtion, 250 U/ml) that was agitated at 37°C. for 45 minutes. The cell suspensions were then poured through a 40 μmcell strainer to isolate cells from scaffold particles and the cellswere pelleted and washed with cold PBS and counted using a Z2 coultercounter (Beckman Coulter). To assess, DC infiltration and activation,subsets of the total cell population isolated from PLG matrices werethen stained with primary antibodies (BD Pharmingen, San Diego, Calif.)conjugated to fluorescent markers to allow for analysis by flowcytometry. APC-conjugated CD11c (dendritic cell marker), FITC-conjugatedMHCII and PE-conjugated CD86 (B7, costimulatory molecule) stains wereconducted for DC recruitment and activation analysis. To furtherdelineate the presence of specific DC subsets, cells were also stainedwith APC-conjugated CD11c and PE-conjugated PDCA-1 (plasmacytoid DCmarker) or APC-conjugated CD11c and PE-conjugated CD8 (CD8 DCs). Toassess T-cell infiltration PE-Cy7 conjugated CD3 stains were performedin conjunction with APC-conjugated CD8a (CD8 T cells), and PE-conjugatedFoxP3 (Treg) and analyzed with flow cytometry. Cells were gatedaccording to single positive FITC, APC and PE stainings and usingisotype controls. The percentage of cells staining positive for eachsurface antigen was recorded.

Tumor Growth Assays, Protective Cytokines and Trp2 Pentamer Analysis

PLG scaffolds with melanoma tumor lysates and GM-CSF in combination withCpG-ODN, MPLA, or P(I:C) were implanted subcutaneously into the lowerleft flank of C57BL/6J mice. For prophylactic vaccinations, animals werechallenged 14 days later with a subcutaneous injection of 10⁵ B16-F10melanoma cells (ATCC, Manassas, N.J.) in the back of the neck. Animalswere monitored for the onset of tumor growth (approximately 1 mm³) andsacrificed for humane reasons when tumors grew to 20-25 mm (longestdiameter).

To assess PLG vaccine efficacy in the therapeutic setting, C57/BL6J micewere challenged with a subcutaneous injection of 5×10⁵ B16-F10 melanomacells (ATCC, Manassas, N.J.) in the back of the neck. At day 9 aftertumor challenge PLG vaccines loaded with 3000 ng GM-CSF in combinationwith 100 μg of CpG-ODN, MPLA or P(I:C), and tumor lysates were implantedsubcutaneously into the lower left flank of C57BL/6J mice. A subset ofmice were vaccinated again 10 days after the initial vaccination (Days19 and 23).

To determine in vivo IL-12p70 concentration at the matrix implant site,adjacent tissue was excised and digested with tissue protein extractionreagent. After centrifugation, the concentrations of IL-12, in thesupernatant were then analyzed with ELISA (R&D systems), according tothe manufacturer's instructions.

To determine the generation of TRP-2-specific cytotoxic T lymphocytes,single cell suspensions were prepared from the spleens of mice immunizedwith PLG vaccines [Antigen+3000 ng GM-CSF+100 μg (CpG or MPLA or P(I:C)]at various timepoints. These cells were initially stained withPE-H-2Kb/TRP2 pentamers (Sigma Aldrich), and subsequently stained withFITC-anti-CD8 and PE-CY7 CD3 mAb (mAb (BD Pharmingen, San Diego) beforebeing analyzed using flow cytommetry.

Tumor Infiltrating Leukocyte (TIL) Characterization

On the indicated days, B16-F10 tumors were removed from mice, anddigested in 1 mg/mL collagenase II (250 U/ml) (Worthington, Lakewood,N.J.) and 0.1 mg/mL DNase for 1 hour at 37° C. Dissociated cells werefiltered through a 40-μm filter, and directly stained with antibodiesfor phenotype characterization by fluorescence-activated cell sorting(FACS) analysis. APC-anti-CD8 and PE-Cy7-anti CD3 were used to identifyT cells isolated from the B16F10 tumors. These TILs were also costainedwith FITC-anti-IFNγ, and PE-anti-CD107a. All antibodies were obtainedfrom eBioscience, San Diego, Calif.

Statistical Analysis

All values in the present study were expressed as mean±S.D. Thesignificant differences between the groups were analyzed by a Student'st test and a P value of less than 0.05 was considered significant.

Controlled GM-CSF and TLR Agonist Presentation

Macroporous, poly-lactide-co-glycolide (PLG) matrices (FIG. 15F) weredesigned to quickly release GM-CSF (Ali et al., 2009 Nat Mater, 2:151-8), approximately 60% of the protein was released by day 10 (FIGS.15A, 15B, and 15E), to induce the recruitment of DCs or theirprecursors. GM-CSF loaded PLG scaffolds were also modified to presentTLR-activating, CpG-ODN, MPLA and P(I:C) molecules, as danger signals.The in vitro release kinetics of GM-CSF were similar in all conditions(FIGS. 15A, 15B, and 15E). TLR agonists were more stably associated withscaffolds, as approximately 20-30% of incorporated CpG-ODN, P(I:C) andMPLA was released over the first 10 days in vitro, followed by slow andsustained release of danger signals over the next 14 days. Presentationof the TLR agonists was designed to provide a long-term, local signal toactivate DCs Importantly, the relatively high molecular weight andcomposition of the particular PLG chosen to fabricate scaffolds resultsin slow scaffold degradation, allowing for long-term analysis of thevaccine site and its regulation over DC activation and T cell immunity.

Controlled DC Generation and Activation In Vivo

To examine the ability of PLG matrices to recruit and activate dendriticcells in vivo, matrices delivering GM-CSF in combination with dangersignals were implanted subcutaneously into the backs of C57BL/6J mice.The magnitude of DC infiltration and activation into the matrices wasdetermined by FACS analysis of cell populations isolated from thepolymeric material after 7 days. Control matrices delivering GM-CSFalone contained 2.41±0.24×10⁵ CD11c(+) (FIGS. 16E and 16F) cells, withrelatively low expression levels of the activation markers, MHCII (2.57%of total CD11c(+) cells) and CD86 (4.58% of CD11c(+) cells). Inclusionof TLR-activating danger signals into PLG matrices dramatically enhanceddendritic cell generation and activation in situ. Presentation ofCpG-ODN, MPLA and P(I:C) enhanced the total number of recruited DCs by2.5, 1.9, and 2.2 fold, respectively (FIG. 16F), as compared to GM-CSFdelivery alone. Analysis of the activation state of matrix-resident DCsrevealed that local TLR induction produces significant percentages ofactivated DCs, as CD11c(+) cells positive for MHCII(+) and CD86(+),comprised approximately 30%, 19%, and 28% of the total cells recruitedto CpG, MPLA and P(I:C) loaded matrices, respectively. Matricespresenting TLR agonists mediated approximately 15 (MPLA) to 20 (P(I:C))to 23-fold (CpG-ODN) increases in the total number of activated DCs atthe implant site relative to control matrices devoid of this signaling(FIG. 16F).

Stimulation of the cells which infiltrated PLG matrices with CpG-ODN,MPLA or P(I:C) enriched the numbers of CD11c(+)PDCA-1(+) pDCs andCD11c(+)CD8(+) cDCs (FIG. 16G) relative to controls. The danger signalsincreased the numbers of pDCs at the implant site by approximately4-fold relative to control matrices, with an average pDC number of140,000 cells residing in the scaffolds presenting any of the TLRagonists (FIG. 16G). CD8(+) DCs were also present at the implant site atapproximately 5-fold higher levels with MPLA and P(I:C) presentation andat a 9-fold higher number when utilizing CpG-ODN as a stimulant.Strikingly, the local delivery of TLR-activating agents promoted thelocal production of IL-12 (200-400 ng/ml) at the implant site (FIG.16H), with CpG and P(I:C) inducing the highest levels. The IL-12concentration correlated with the increased numbers of activated DCs andDC subsets in these conditions (FIG. 16H). Additionally, theconcentrations of a panel of candidate inflammatory cytokines wereassayed at the vaccine site. Similar elevated levels of IFN-α (FIG. 21A)resulted from CpG-ODN and P(I:C) induction while MPLA had no effect onIFN-α concentration. However, MPLA led to 4-fold higher levels of TNF-α(FIG. 21B). IL-12 concentrations at MPLA loaded matrices were 2-foldlower than CpG-ODN and P(I:C). TNF-α inhibits monocyte and DC derivedIFN-γ, IL-12, and T cell priming (Hodge-Dufour et al., 1998 Proc. Natl.Acad. Sci. USA, 95: 13806-13811) and the aforementioned cytokineprofiles, suggesting that MPLA loaded matrices are less efficient atstimulating anti-tumor T cell responses compared to matricesincorporating CpG-ODN and P(I:C) signaling.

Prophylactic Vaccination and Correlates to its Efficacy

Since PLG matrices presenting TLR agonists generate distinct andactivated DC populations in situ and potent cytokine production, theanti-tumor efficacy of these systems were tested in the poorlyimmunogenic, B16-F10 melanoma model. B16 tumor lysates were used to actas a source of tumor antigen in vaccine formulations. ProphylacticPLG-based vaccines presenting both B16-F10 tumor lysates and GM-CSFresulted in 10% of the vaccinated mice surviving, tumor-free (FIG. 17A),after an otherwise lethal cell challenge at day 14 post-vaccinationImportantly, antigen loaded matrices with GM-CSF in combination with TLRagonists produced significant, and long-term tumor protection. CpG-ODN,MPLA and P(I:C) presentation from PLG vaccines resulted in 90, 80 and70% survival rates (FIG. 19B). Regression analysis was subsequentlyperformed to determine whether induction of long-term survival wasrelated to pDC, CD8(+), and IL-12 levels at the vaccine site: previouslypublished data using various doses of CpG-ODN were included in theanalysis. Strikingly, animal survival rates were strongly correlatedwith the numbers of pDCs, CD8(+) DCs and with endogenous IL-12 generatedby PLG vaccines (FIGS. 17A-C). These findings demonstrate the importanceof antigen cross-presentation by CD8(+) DCs and the Th1-promotingcytokine, IL-12 to vaccine efficacy.

Therapeutic Vaccination and Anti-Tumor T Cell Activity

As specific vaccine formulations containing various TLR agonistsproduced significant numbers of activated DCs and conferred prophylacticimmunity, studies were carried out to determine whether the vaccineswould lead to superior therapeutic responses and cytotoxic T-cellresponses. Mice challenged with 5×10⁵B16-F10 melanoma cells weresubsequently vaccinated at days 9 and 19, after tumors were established.All tumor-bearing mice implanted with control PLG matrices demonstratedrapid tumor growth and required euthanasia by Day 24, as expected (FIGS.19A and B) PLG vaccines presenting MPLA as an adjuvant decreased therate of tumor progression (FIG. 19A), and a slight increase in meansurvival time (˜1.5 fold increase) over controls was found (FIGS. 19Aand B). Complete tumor regression (Tumors <36 mm²) and long termsurvival of mice (33% survival) was achieved in the subset of micevaccinated with PLG vaccines exploiting P(I:C) and CpG-ODN as anadjuvant.

FACs analysis was used to characterize the numbers of B16-F10tumor-infiltrating leukocytes (TILs) induced by the various vaccines.Significantly greater numbers of CD8(+) CTLs per 1×10⁶ tumor cells werepresent in animals treated with TLR-agonist-loaded vaccines, as comparedwith control animals (FIGS. 19C-D). CD8(+) T cell infiltrates werefurther characterized for IFNγ expression and expression of CD107a, amarker for cytotoxic-associated cell degranulation. These cellpopulations were markedly enhanced in vaccine treated animals (FIGS.19C-D). Vaccines featuring CpG-ODN, P(I:C) and MPLA signaling resultedin approximately 6.1, 3.1 and 1.4 fold increases in IFNγ(+), CD107a(+)TILs in comparison to controls. Moreover, CpG loaded vaccines resultedin significantly higher numbers of activated TILs in comparison to theirP(I:C) and MPLA counterparts (FIG. 19D).

The activation of systemic CTL responses was also monitored by stainingsplenocytes with MHC class I/TRP2 peptide pentamers to identify CTLswith specificity to tyrosinase-related protein (TRP)-2. This is a mainantigenic target of melanoma vaccines in mice and humans A significantexpansion of TRP2 specific CTLs was observed in the spleens of micevaccinated with CpG-ODN, MPLA and P(I:C) loaded vaccines, in comparisonto controls devoid of TLR agonists (FIG. 19E). Taken together, thesedata indicate that vaccine formulations containing various TLR agonistsproduce significant and systemic anti-melanoma CTLs in correlation withthe activation of specific DC subsets and reduce tumor burden.

Vaccine Efficacy is Impaired in Mice Lacking CD8(+) DCs

Since PLG vaccines incorporating TLR agonists were capable of generatingCD8(+) DC populations in situ, which correlated to potent anti-tumor CTLresponses and survival (FIGS. 17A-D and 19A-F), studies were carried outto examine whether these cells were required to confer anti-tumorimmunity in vivo. Batf3−/− transgenic mice were used in theseexperiments, as lack CD8(+) DCs, without exhibiting abnormalities inother hematopoietic cell types or tissue architecture (Hildner et al.,2008 Science, 322: 1097-1100). Wild-type and Batf3−/− mice werevaccinated with CpG-ODN loaded PLG vaccines and challenged with B16-F10cells 14 days later. Vaccination of wildtype mice promoted completeprotection against tumor growth and long-term survival (100% survival),as expected, but vaccinated Batf3−/− were not protected and tumor growthrates were similar to untreated, wild-type animals (FIG. 20A). Moreover,vaccinated Batf3−/− failed to produce the local CTL responses observedin wild-type mice, and a 3-fold decrease in TRP2 specific cytotoxic Tcells coincided with higher ratios of FoxP3(+) T regulatory (Tregs)cells at the vaccine site in this condition (FIG. 20B). These resultsindicated that a lack of CD8(+) DCs resulted in limited cytotoxicity andallowed regulatory pathways mediated by Tregs to potentially extinguishthe vaccine mediated, immune response.

Wild type mice were also able to induce the production of the T cellgrowth factor, IL-12, at the vaccine site at 5-fold higher levels thanfound in vaccinated Batf3−/− mice (FIG. 20C). The partial loss of IL-12production in CD8(+) DC knock-out mice indicates that these cells areimportant producers or mediators of this Th1-polarizing cytokine. Thevaccine sites of CD8(+) T cell knock out mice also showed decreasedlevels of IL-12 and IFN-γ (FIGS. 21C and D). The IL-12-IFNγ pathway is apositive feedback mechanism with each cytokine augmenting production ofits counterpart. The data with Batf3−/− mice and CD8(+) T cell knock-outmice indicate that the immune response resulting from vaccination innormal mice may be amplified by cytokine mediated cross talk between DCsand CTLs. Finally, the systemic production of anti-tumor CTLs was alsoimpaired in Batf3−/−, as a 3 fold reduction in Trp2 specific CTLs wasmeasured in the spleens of these mice in comparison to wild-typecontrols (FIG. 20D). These results indicate that vaccine efficacy inthis system is critically regulated by CD8 DCs, via their ability tocross present tumor antigens, to produce Th-1 induction factors such asIL-12, and to generate and interact with CTLs.

To address the limitations of earlier cancer vaccines, a PLG matrix wasutilized that controls the presentation of tumor lysate, GM-CSF and TLRagonists to create a vaccine node that recruits and activates multipleDC subsets in situ. The contribution of DC subsets to vaccine efficacywas analyzed and it was demonstrated that effective tumor cell killingrequired the participation of CD8(+) DCs, with strong correlations topDCs and IL-12. These components were critical to the vaccine resultsregardless of the type or dose of stimulant incorporated within thescaffolds.

Inclusion of TLR agonists was required to activate DCs, in general,increasing their surface expression of MHCII and the costimulatorymolecule, CD86 (FIGS. 16A-H) indicating an enhanced capacity to presentantigen and activate T cell populations. In particular, appropriate TLRsignaling enhanced the generation of CD8(+) and pDC subsets at thevaccine site and stimulated the production of IFNs and the potent T cellgrowth factor, IL-12. Moreover, removal of TLR agonists from the systemresulted in decreased numbers of Trp2 specific, cytotoxic CD8(+) T cellslocally at the vaccine site and systemically in spleens and tumors andthis coincided with reduced survival in vaccine studies. PLG vaccinespresenting P(I:C) or CpG-ODN induced potent tumor rejection intherapeutic models of B16-F10 melanoma, causing complete tumorregression in over a third of vaccinated animals and eradicating tumorsreaching 35 mm² in size. Analysis of the tumor sites in vaccinatedanimals demonstrated intense and activated CD8(+), cytotoxic T cellinfiltrates likely effecting tumor-cell killing. These systemsoutperformed the preclinical results of current vaccine modalitiesextensively studied in the clinic, including irradiated tumor cellvaccines and DC based vaccines (Gilboa E, 2007 J Clin Invest, 117:1195-1203; Banchereau J. and Steinman R M 2007 Nature, 49: 419-426; Aliet al., 2009 Sci Transl Med, 1: 8-19; Rosenberg et al., 2004 Nat Med,10: 909-915; Klebanoff et al., 2006 Immunol Rev, 211: 214-224). Vaccinespresenting MPLA signaling also slowed tumor growth rates, but did notcause tumors to completely regress. This may be explained by the factthat, CpG-ODN and P(I:C) signaling outperformed MPLA in terms ofpromoting higher average levels of cell surface activation markers onDCs and cytokine profiles that promote Th-1 polarization and CTLresponses. Additionally, MPLA was a strong inducer of TNF-α, incomparison to CPG-ODN and P(I:C), which can inhibit IL-12, and IFNpathways priming CTL responses (Hodge-Dufour et al., 1998 Proc. Natl.Acad. Sci. USA, 95: 13806-13811). Thus, the differential effects of TLRagonists on survival rates are consistent with the numbers and subsetsof activated DCs produced at the vaccine site and cytotoxic T-cellactivity. Combinations of TLR agonists, e.g., poly (I:C) and CpG or poly(I:C) and MPLA, lead to a synergistic anti-tumor effect.

All vaccinated mice without the CD8(+) DC compartment, Batf3−/− mice,generated tumors in prophylactic models that produced 90% tumor-freesurvival rates in wild-type mice. Cytokine analysis of the vaccine siteof these animals revealed that local IL-12 levels, and CTL responseswere markedly reduced indicating that CD8(+) DCs are an important sourceof IL-12, and Th-1 polarization. Without CD8(+) DC participation,vaccination not only resulted in reduced cytotoxic, CD8(+) T cellactivity (at tumor site and spleen) it also allowed the progression ofTreg activity. High FoxP3(+) Treg to CD8(+) T-cell ratios indicatesunbalanced immunosuppression that extinguishes vaccine efficacy andpromotes tumor growth (Quezada et al., 2006 J. Clin. Invest., 116:1935-1945; Hodi et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105:3005-3010). CD8(+) DCs and IL-12 can cause Tregs inhibition or theirconversion to IFNγ-producing, effector T cells (Wei et al., 2009Immunity, 30: 155-167; Zhou et al., 2009 Nat Immunol., 10: 1000-1007;Oldenhove et al., 2009 Immunity, 31: 772-786), and these mechanisms arepotentially critical to the efficacy of material-based cancer vaccines.

An interesting aspect of these systems is CTL homing to the vaccine sitedue to long term antigen presentation, and knocking out CD8(+) T cellsresulted in a significant reduction of local levels of IFNγ and IL-12(S3 &S4). T cell derived IFNγ enhances DC expression of IL-12 andcostimulatory molecules creating a feedback loop that amplifiesCTL-mediated responses to infection. In this setting, after vaccinepriming, T cells that home back to the antigen-presenting vaccine sitemay be important vaccine components themselves as they may sustain andamplify CTL responses via IFNγ-mediated DC activation and IL-12production. These findings provide evidence that CD8(+) DCs, pDCs andIL-12, or their equivalent functionally (i.e., appropriate antigenpresentation, and Th1 polarization) lead to improved material-basedcancer vaccines. The methods and devices described herein are alsouseful for other clinical indications, such as infectious and autoimmunedisease.

As described above, three different types of pathogen associatedmolecular patterns (PAMPs) were incorporated into or onto structuralpolymeric devices such as PLG disc structures/scaffolds to act asadjuvants in vaccines (3 types; a short oligonucleotide (CpG-ODN); asynthetic RNA—(Poly(I:C); P(I:C)), a synthetic lipid (monophosphoryllipid A; MPLA) (FIGS. 15A-D). Such vaccine formulations recruit andactivate dendritic cells in situ, which was quantitatively assessed(FIGS. 16A-D).

Vaccine-dependent survival in an aggressive melanoma cancer modelcorrelates strongly with the ability of the vaccine to specificallyactivate 2 subsets of dendritic cells—CD8(+) DCs and plasmacytoidDCs—regardless of the adjuvant utilized in the vaccine system (FIGS.17A-C). This correlation has been confirmed utilizing 4 differentvaccine adjuvants in the PLG vaccine. These vaccines induce potent tumorrejection in a therapeutic model of melanoma, by activating specific Tcell responses that have been detected at the vaccine site and at tumors(FIGS. 18A-D and 19A-D). These findings demonstrate the PLG vaccinesystem's versatility in incorporating different types of agonists thatstimulate different pathways in innate and adaptive immune responses(FIGS. 15A-D).

DC subsets that are critical for anti-tumor immune responses wereidentified. Subsets of DCs include myeloid dendritic cell (mDC),plasmacytoid DCs, and CD8+ DCs. mDCs are most similar to monocytes. mDCare made up of at least two subsets: (1) the more common mDC-1, which isa major stimulator of T cells; and (2) the extremely rare mDC-2, whichmay have a function in fighting wound infection. mDCs secrete IL-12 andare characterized by TLR 2 and TLR4. Plasmacytoid DCs look like plasmacells, but have certain characteristics similar to myeloid dendriticcells, can produce high amounts of interferon-alpha, and arecharacterized by TLR7 and TLR9. The TLR agonist, CpG, binds to TLR9.CD8+ DCs in mice are equivalent to CD141+ dendritic cells.

CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, andblood. They are characterized by high expression of toll-like receptor 3(TLR3), production of IL-12p70 and IFN-β, and superior capacity toinduce T helper 1 cell responses, when compared with the more commonlystudied CD1c+DC subset. Polyinosine-polycytidylic acid (polyI:C)-activated CD141+ DCs have a superior capacity to cross-presentantigens to CD8+ cytotoxic T lymphocytes than poly I:C-activated CD1c+DCs. Thus CD141+DC subset represents an important functionally distincthuman DC subtype with characteristics similar to those of the mouseCD8α+DC subset. CD141+ DCs play a role in the induction of cytotoxic Tlymphocyte responses and their activation is important for vaccinationagainst cancers, viruses, and other pathogens.

CD141+ DCs and plasmacytoid DCs are critical for successful cancervaccination (prophylactic and therapeutic). The results described hereinindicate that p(I:C) in vaccine device stimulates CD141+ DCs in humans(CD8+ DCs in mice) and CpG stimulates plasmacytoid DCs. Devices with oneor both of these TLR agonists lead to potent DC activation and thegeneration of significant prophylactic and therapeutic anti-tumor immuneresponses. A combination of different TLR agonists, e.g., a combinationof p(I:C) and CpG, in a device leads to a synergistic effect in theactivation of a DC immune response against tumors. CD141+ DCs andplasmacytoid DCs have not been specifically utilized or targeted incurrent clinical trials. The data obtained from these experiments wasused to design cancer vaccine systems and provide a more informedtranslation of immune response data from mouse to humans.

Synergistic Effect of P(I:C)+CpG ODN on Tumor Inhibition

FIG. 22 shows the results of a vaccine experiment where P(I:C) andCpG-ODN in combination were examined versus the CpG-ODN and P(I:C)alone. As shown in FIG. 22, the combination of the TLR agonists P(I:C)and CpG-ODN outperformed the vaccines incorporating CpG-ODN or P(I:C)alone. Specifically, PLG vaccines incorporating CpG-ODN and P(I:C) actsynergistically to generate significant tumor inhibition, reduced tumorburden, and to generate improved anti-tumor immune responses.

FIG. 22 shows the overall survival of mice bearing melanoma tumors, andtreated with either blank matrices [Blank] or matrices loaded withCpG-ODN or P(I:C) alone or in combination [CpG-ODN+P(I:C)] (n=8). Micewere challenged with 5×10⁵ B16-F10 cells and vaccinated 3 days laterwith PLG vaccines. Total dose of TLR agonist was approximately 100 μg inall vaccines.

Example 8 Inflammatory Cytokines Presented from Polymer MatricesDifferentially Generate and Activate DCs In Situ

During infection, inflammatory cytokines mobilize and activate dendriticcells (DCs), which are essential for efficacious T cell priming andimmune responses that clear the infection. Described herein is thedesign of macroporous poly(lactide-co-glycolide) (PLG) matrices thatrelease the inflammatory cytokines GM-CSF, Flt3L and CCL20, in order tomimic infection-induced DC recruitment. The ability of these infectionmimics to function as cancer vaccines was examined via induction ofspecific, anti-tumor T cell responses. As described in detail below, allvaccine systems tested were able to confer specific anti-tumor T cellresponses and long-term survival in a therapeutic, B16-F10 melanomamodel. However, GM-CSF and Flt3L vaccines resulted in similar survivalrates, and outperformed CCL20 loaded scaffolds, even though they haddifferential effects on DC recruitment and generation. GM-CSF signalingwas identified as the most potent chemotactic factor for conventionalDCs and significantly enhanced surface expression of MHC(II) andCD86(+), which are utilized for priming T cell immunity. In contrast,Flt3L vaccines led to greater numbers of plasmacytoid DCs (pDCs),correlating with increased levels of T cell priming cytokines thatamplify T cell responses. These results demonstrate that 3D polymermatrices modified to present inflammatory cytokines are utilized toeffectively mobilize and activate different DC subsets in vivo forimmunotherapy.

An exemplary amino acid sequence of human Flt3 is provided below(GenBank Accession No.: P49771.1 (GI:1706818), incorporated herein byreference; SEQ ID NO: 12):

  1 mtvlapawsp ttylllllll ssglsgtqdc sfqhspissd favkirelsd yllqdypvtv 61 asnlqdeelc gglwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl121 rfvqtnisrl lqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleatapt181 apqpplllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

An exemplary amino acid sequence of human CCL20 is provided below(GenBank Accession No.: AAH20698.1 (GI:18088857), incorporated herein byreference; SEQ ID NO: 11):

 1 mcctksllla almsvlllhl cgeseasnfd cclqytdril hpkfivgftr qlanegcdin61 aiifhtkkkl svcanpkqtw vkyivrllsk kvknm

Materials & Methods Mice

C57BL/6 mice (6-8-week-old female; Jackson Laboratories) were used inthe experiments described in Example 8.

Primary Cells (DCs) Isolation and Culture

Primary bone-marrow-derived dendritic cells (BMDCs) were flushed fromthe femurs of C57BL/6 mice and cultured in 100-mm bacteriological petridishes (Falcon number 1029/Becton Dickinson). Cell culture mediumRPMI-1640 (R10) (Sigma) was supplemented with 1% Penicillin-Streptomycin(Invitrogen), 2 mM 1-Glutamine (Invitrogen), 50 μM 2-mercaptoethanol(Sigma) and 10% heat-inactivated fetal bovine serum (FBS, Invitrogen).At day 0, bone marrow leukocytes were seeded at 2×10⁶ cells per 100-mmdish in 10 ml R10 medium containing 20 ng/ml granulocyte-macrophagecolony-stimulating factor (GM-CSF) (Peprotech). At day 3, another 10 mlR10 medium containing 20 ng/mL GM-CSF was added to the plates. At days 6and 8, half of the culture supernatant was collected and centrifuged,the cell pellet was resuspended in 10 ml fresh R10 containing 20 ng/mLGM-CSF, and placed back into the original plate. The non-adherent cellpopulation in the culture supernatant was used between days 8 and 12 forall the experiments.

Transwell Migration Studies for Chemotaxis and Chemokinesis

Transwell migration studies were performed by plating hone marrowderived dendritic cells in the top well of 6.5 mm transwell dishes(Costar, Cambridge, Mass.) with a pore size of 5 μm. The chemotacticeffects of GM-CSF, FL (Flt3), and CCL20 on the migration of BMDCs wasassessed by placing 500 ng/ml of recombinant murine GM-CSF, FL or CCL20(Peprotech, Rocky Hill, N.J.) in the bottom wells and 3×10⁵ DCs in thetop wells. For chemokinesis studies, the concentration of cytokine ineach compartment was equal at 500 rig/ml and 3×10⁵ DCs were added to thetop wells. The number of cells that migrated from the top well to thebottom well through the porous membrane was counted at the end of 12 hto quantify migration. Cells that had migrated to the bottom well werecollected by treatment with 0.25% trypsin-0.03%ethylenediaminetetraacetic acid (EDTA, Invitrogen) and counted with a Z2coulter counter (Beckman Coulter, Inc).

Matrix Fabrication

A 85:15, 120 kD copolymer of D,L-lactide and glycolide (PLG) (Alkermes,Cambridge, Mass.) was utilized in a gas-foaming process to form porousPLG matrices. PLG microspheres encapsulating GM-CSF, FLt3L, or CCL20were first made using standard double emulsion, incorporatingapproximately 170 ng/mg of protein PLG microspheres. PLG microsphereswere then mixed with 150 mg of the porogen, sucrose (sieved to aparticle size between 250 μm and 425 μm), and compression molded. Theresulting disc was allowed to equilibrate within a high-pressure CO₂environment, and a rapid reduction in pressure causes the polymerparticles to expand and fuse into an interconnected structure. Thesucrose was leached from the scaffolds by immersion in water yieldingscaffolds that were 90% porous.

To incorporate tumor lysates into PLG scaffolds, biopsies of B16-F10tumors that had grown subcutaneously in the backs of C57BL/6J mice(Jackson Laboratory, Bar Harbor Me.), were digested in collagenase (250U/ml) (Worthington, Lakewood, N.J.) and suspended at a concentrationequivalent to 10⁷ cells per ml after filtration through 40 μm cellstrainers. The tumor cell suspension was subjected to 4 cycles of rapidfreeze in liquid nitrogen and thaw (37° C.) and then centrifuged at 400rpm for 10 min. The supernatant (1 ml) containing tumor lysates wascollected, incubated with the PLG microspheres and lyophilized and theresulting mixture was used to make PLG scaffold-based cancer vaccines.

To incorporate CpG-ODNs into PLG scaffolds, CpG-ODN 1826, 5′-tcc atg acgttc ctg acg tt-3′, (Invivogen, San Diego, Calif.; SEQ ID NO: 10) wasfirst condensed with poly(ethylenimine) (PEI, Mn—60,000, Sigma Aldrich)molecules by dropping ODN-1826 solutions into PEI solution, whilevortexing the mixture. The charge ratio between PEI and CpG-ODN(NH3+:PO4−) was kept constant at 7 during condensation. The condensatesolutions were then vortexed with 60 μl of 50% (wt/vol) sucrosesolution, lyophilized and mixed with dry sucrose to a final weight of150 mg. The sucrose containing PEI-CpG-ODN condensate was then mixedwith blank, GM-CSF and/or tumor lysate loaded PLG microspheres to makePLG cancer vaccines.

In Situ Identification of DCs and T Cells

GM-CSF loaded PLG matrices containing approximately 3 μg of GM-CSF,Flt3L, or CCL20 in combination with 100 μg of CpG-ODN were implantedinto subcutaneous pockets on the back of 7-9 week old male C57BL/6Jmice. To analyze DC recruitment by FACS analysis, scaffolds were excisedand the ingrown tissue was digested into single cell suspensions using acollagenase solution (Worthingtion, 250 U/ml) that was agitated at 37°C. for 45 minutes. The cell suspensions were then poured through a 40 μmcell strainer to isolate cells from scaffold particles and the cellswere pelleted and washed with cold PBS and counted using a Z2 coultercounter (Beckman Coulter). To assess, DC infiltration and activation,subsets of the total cell population isolated from PLG matrices werethen stained with primary antibodies (BD Pharmingen, San Diego, Calif.)conjugated to fluorescent markers to allow for analysis by flowcytometry. APC-conjugated CD11c (dendritic cell marker), FITC-conjugatedMHCII and PE-conjugated CD86 (B7, costimulatory molecule) stains wereconducted for DC recruitment and activation analysis. To delineate thepresence of the plasmacytoid DC subset, cells were also stained withAPC-conjugated CD11c and PE-conjugated PDCA-1 (plasmacytoid DC marker).Cells were gated according to single positive FITC, APC and PE stainingsusing isotype controls. The percentage of cells staining positive foreach surface antigen was recorded.

For the immunostaining of DC infiltrates in paraffin sections ofscaffolds, samples were prepared and re-hydrated according to standardprocedures. Antigen retrieval was performed with citrate buffer in apressurized cooker (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0, for 5min in 950 C). After brief washes with PBST buffer (0.01% Tween-20),samples were blocked with 5% goat serum in staining buffer (5% BSA, 2%Fetal Bovine Serum in PBST, pH 7.4) for 1 hour in ambient temperature.Anti-CD11c Armenian Hamster IgG (20 mg/ml, Abcam, Cambridge, Mass.) wasdiluted in staining buffer and allowed to bind overnight at 40 C in ahumidified chamber. After washing 3 times with PBST, Alexa®594 Goatanti-Hamster (5 mg/ml, Life Technologies, Grand Island, N.Y.) secondaryIgG was diluted in staining buffer and applied for 1 h at RT. Afterthree washes with PBS, samples were air-dried and mounted with ProLongGold Antifade reagent with DAPI for nuclear staining (Life Technologies,Grand Island, N.Y.). Confocal tile scans were obtained and processedwith Zeiss LSM 710 laser-scanning microscope and bundled software(Zeiss, Thornwood, N.Y.).

Tumor Growth Assays, Protective Cytokines and Trp2 Pentamer Analysis

PLG scaffolds containing B16-F10 melanoma tumor lysates, 100 μg CpG-ODNin combination with 3 μg GM-CSF, Flt3L or CCL20 were implantedsubcutaneously into the lower left flank of C57BL/6J mice to act ascancer vaccines. To assess PLG vaccine efficacy in the therapeuticsetting, C57/BL6J mice were challenged with a subcutaneous injection of5×10⁵ B16-F10 melanoma cells (ATCC, Manassas, N.J.) in the back of theneck, and 3 days after tumor challenge PLG vaccines were implantedsubcutaneously into the lower left flank. Animals were monitored for theonset of tumor growth (approximately 1 mm³) and sacrificed for humanereasons when tumors grew to 20-25 mm (longest diameter).

To determine the in vivo IL-12p70 and IFN-γ concentration at the matriximplant site, adjacent tissue was excised and digested with tissueprotein extraction reagent at Day 10 after implantation (Pierce). Aftercentrifugation, the concentrations of IL-12 and IFN-γ, in thesupernatant were then analyzed with ELISA (R&D systems), according tothe manufacturer's instructions. To determine the generation ofTRP-2-specific cytotoxic T lymphocytes, single cell suspensions wereprepared at Day 10 from the spleens of mice immunized with PLG vaccines.These cells were initially stained with PE-H-2Kb/TRP2 pentamers (SigmaAldrich), and subsequently stained with FITC-anti-CD8 and PE-CY7 CD3 mAb(mAb (BD Pharmingen, San Diego) before being analyzed using flowcytometry.

The Role of Dendritic Cells in the Immune Response

Dendritic cells (DCs) orchestrate immune responses to infection andtumors by priming and propagating specific, cytotoxic T lymphocyte (CTL)responses. Immature DCs residing in peripheral tissue detect foreignsubstances (i.e. antigens) unique to invading pathogens, and areactivated by stimuli, such as pathogen associated molecular patterns(PAMPs) or products of dying cells (i.e. “danger signals”), originatingduring pathogen induced inflammatory responses. Maturing DCs mature bothprocess and present antigens on major histocompatibility complexes (MHC)receptors, and express the costimulatory molecules CD80 and CD86, bothof which are required for effector T-cell stimulation. Another importantresult of DC maturation by ‘danger signaling’, is that DCs acquire theability to home to the lymph nodes to engage and activate naive T-cells,enabling the T cells to recognize the antigens DCs are presenting.

The ability of particular DCs to initiate and control immune responsesis a consequence of both their localization within tissues and theirspecialized capacity for mobilization. DCs originate from pluripotentstem cells in the bone marrow, enter the blood stream and localize intoalmost all organs. Based on the relative expression of a series ofsurface markers, different subsets of DCs or DC precursors can beidentified in peripheral blood, including plasmacytoid DCs (pDCs) andconventional DCs (cDCs)2. pDCs are major type I interferon (IFN)producers, and specialize in activating adaptive immune responses tovirus challenge via cytokine signaling. CD11c(+) cDCs, such as epidermalDCs, are especially adept at antigen presentation and co-stimulation ofT cells.

Upon microbial invasion and inflammation, DCs rapidly migrate into thedraining lymph nodes and primary sites of infection at rates that vastlyoutnumber other APCs, such as macrophages. The production of most DCsubsets, including (pDCs) is controlled in the steady state by thecytokine Fms-related tyrosine kinase 3 ligand (FL). Other cytokines,such as GM-CSF and CCL20, released by damaged or infected cells,actively recruit and localize cDCs to the sites of inflammation. Ininflammatory models, both in vivo and in vitro, these inflammatorycytokines have been shown to also enhance DC migration and proliferationand may regulate DC activation state. The quantity of DCs activatedduring infection or within tumors is correlated with the strength of thesubsequent immune response and disease prognosis.

To generate sufficient numbers of dendritic cells (DCs) forimmunotherapy, laboratory-based culture of DC precursors withinflammatory cytokines, such as granulocyte macrophage-colonystimulating factor (GM-CSF) and FL (Flt3) has often been used. DCsmodified in vitro to present tumor antigens are capable of elicitingantitumor effects in murine models upon transplantation. Initialclinical testing of ex vivo DC-based vaccines has revealed the inductionof tumor regression in a subset of cancer patients, but little survivalbenefit. Protocols involving the ex vivo manipulation of DCs are limitedby the quantities and types of DCs that can be produced, poorengraftment efficiency and LN homing, and loss of DC activation uponinjection in the in vivo environment.

To address these limitations, infection-mimicking materials weredeveloped to present inflammatory cytokines in combination with a dangersignal to recruit and activate DCs in vivo. As described below, theabilities of multiple inflammatory cytokines, GM-CSF, FL (Flt3), andCCL20 to recruit and activate DCs when delivered from macroporous,implantable polymer scaffolds was examined. Also, nanoparticlescontaining cytosine-guanosine (CpG) rich oligonucleotide (CpG-ODN)sequences were immobilized onto scaffolds, as CpG-ODN are expressed inbacterial DNA, and are potent danger signals that can stimulateactivation of matrix resident DCs. Finally, the ability of these systemsto prime anti-tumor T cell responses and confer tumor protection viapresentation of cancer antigens was examined

In Vitro Chemotaxis and Chemokinesis of Cytokines

In vitro transwell studies were conducted to investigate the chemotaxisand chemokinesis effects of bone-marrow derived DCs in response toGM-CSF, FL and CCL20. GM-CSF gradients promoted significant DCchemotaxis, as DC migration in this condition was approximately 50%higher than the control condition (FIG. 23A). Similar effects onchemokinesis were observed, as GMCSF exposure promoted a similar 50%increase in the number of migrated cells in response to homogenouslevels of the cytokine (FIG. 23B). In contrast, FL and CCL20 had noeffect on the chemotaxis or chemokineses of DCs in these assays (FIGS.23A-B). These results indicate that GM-CSF has a superior effect on DCmobilization and recruitment in comparison to FL and CCL20.

Controlled Release of Cytokines and In Vivo DC Recruitment

Macroporous, poly-lactide-co-glycolide (PLG) matrices were designed toprovide long-term and sustained release of GM-CSF, FL, and CCL20 (FIG.23A) and to house DCs for activation. These PLG scaffolds were 80-90%porous with an average pore size between 125-200 μm to facilitatedendritic cell infiltration. The in vitro release kinetics for the threecytokines were similar, as the matrices quickly released protein with aburst over the first 5 days followed by sustained release over the nextseveral weeks (FIG. 24A). The scaffolds released approximately 43, 36,and 26% of the incorporated GMCSF, FL and CCL20, respectively, by day 4followed by approximately 0.9% daily release of protein over the next 23days (FIG. 24A).

To examine the ability of PLG matrices loaded with inflammatorycytokines to recruit and activate dendritic cells in vivo, PLG matricesdelivering GM-CSF, FL and CCL20 were implanted subcutaneously into thebacks of C57BL/6J mice and removed at day 7 after implantationImmunohistochemical analysis revealed intense CD11c(+) DC infiltratespenetrating the porous network of all the scaffolds releasing cytokines,and GM-CSF mediated the most dense DC clustering (FIG. 24B). Themagnitude of DC infiltration and activation into the matrices wasquantified by FACS analysis of cell populations isolated from thepolymeric material. Blank PLG matrices recruited approximately 190,000CD11c(+) DCs, whereas scaffolds delivering GM-CSF recruitedapproximately 960,000 DCs, equating to over a 5-fold difference in cellrecruitment (FIG. 24C). Scaffolds presenting FL and CCL20 recruited 2.5fold more DCs than control conditions, but significantly less thanGM-CSF presenting scaffolds (FIG. 24C). These results are consistentwith the in vitro results that identified GM-CSF as the most potentmobilizing and chemotactic factor for DCs, in comparison to FL andCCL20.

In Vivo DC Activation

PLG scaffolds were modified to present nanoparticles containingTLR-activating, CpG-ODN, as an infection-mimicking danger signal inconcert with delivery with inflammatory cytokines. This dramaticallyenhanced DC activation in situ over control conditions lacking cytokinesignaling (FIG. 25A). Analysis of the activation state ofmatrix-resident DCs revealed that GM-CSF induced recruitment incombination with CpG-ODN produced significant percentages of activatedDCs, as MHCII(+) and CD86(+) DCs comprised approximately 54-66% of thetotal DCs recruited to scaffolds. Approximately 8-fold, 4-fold, and4-fold increases in the total number of activated DCs were found withGM-CSF, at the implant site relative to control matrices devoid ofcytokines (FIG. 25B).

FL presentation in combination with CpG-ODN enriched the PLG matrix withthe highest average number of CD11c(+)PDCA-1(+) pDCs (FIG. 1C),generating over 160,000 resident pDCs. Strikingly, approximately 22% ofthe total cells resident in these scaffolds consisted of this DC subset(FIG. 25A). This indicates that FL is a strong mobilizing agent forpDCs. Scaffolds presenting GM-CSF and CCL20 also significantly enhancedpDC generation, leading to approximately 110,000 resident pDCs. The pDCsubset has been associated with the induction of t-helper 1 (Th1)immunity via its capacity to induce IL-12 and type-1 interferons (IFNs),which are critical to propagating CTL responses to infections andtumors. These data indicate that while the three cytokines tested allenhanced DC recruitment and activation, GM-CSF signaling in combinationwith CpG-ODN produced the highest numbers of activated cDCs, while FLled to the greatest number of resident pDCs.

Induction of T Cell Immunity and Therapeutic Vaccination

B16-F10 tumor lysates were incorporated into PLG matrices as tumorantigens. IL-12 and IFN-γ secretion by activated DCs can primeCTL-mediated immune responses and tumor cell death. Thus, the presenceof these Th-1 inducers at the scaffold implant site was quantified. PLGvaccines presenting FL in combination with CpG-ODN enhanced the localconcentration of IL-12 and IFN-γ by 8 and 13-fold, respectively, overcontrol scaffolds (FIGS. 26A and 26B). GM-CSF release resulted inapproximately 3 and 6-fold increases in the local concentration of IL-12and IFN-γ (FIGS. 26A and 26B). CCL20 release from PLG vaccines led to a2-fold increase in IL-12 concentration, and no effect on IFN-γ levels atthe vaccine site (FIGS. 26A and 26B).

The activation of systemic CTL responses was monitored by stainingisolated splenocytes with MHC class I/TRP2 peptide pentamers. Thisallows one to identify CTLs with specificity to tyrosinase-relatedprotein (TRP)-2, which is a main antigenic target of melanoma vaccinesin mice and humans. A significant expansion of TRP2 specific CTLs wasobserved in the spleens of mice vaccinated with scaffolds incorporatingall three cytokines (FIG. 26C). Vaccines incorporating FL and GMCSF ledto approximately 5 and 4-fold higher numbers of Trp2 specific CTLs thancontrol vaccines, which was a significantly greater effect than foundwith CCL20 vaccines (FIG. 26C). Taken together, these data indicate thatvaccine formulations containing various inflammatory agents are capableof producing significant and systemic anti-melanoma CTLs and the localproduction of Th1 cytokines.

The anti-tumor efficacy of these vaccines were then tested in the poorlyimmunogenic, B16-F10 melanoma, model. C57BL/6J mice were challenged with10⁵ tumor cells, and then vaccinated with PLG vaccines three days later.Animals treated with control scaffolds required euthanization after 30days due to progressive disease. PLG vaccines loaded with cytokinesinduced long-term tumor protection in a significant subset of animals(FIG. 26D). GMCSF, FL and CCL20 presentation from PLG vaccines resultedin 88, 75 and 62% long-term survival rates (FIG. 26D).

Controlled mobilization and activation of DCs and DC precursors is ofparticular interest in the development of ex vivo DC based vaccines, andmore generally the design of material systems that activate the immunesystem in vivo. As described herein, polymers which mimic key aspects ofmicrobial infection effectively recruit DCs for cancer vaccination. PLGscaffolds engineered to release GM-CSF, FL, and CCL20 led to significantnumbers of resident DCs, and the co-presentation of danger signals ledto DC maturation. Even though all vaccine formulations were capable ofinducing tumor protection in a therapeutic model of B16-F10 melanoma,GM-CSF and FL vaccines produced more antigen specific CTLs, higherlevels of Th1 priming cytokines, and greater survival rates whencompared to CCL20 (FIG. 26D).

While GM and FL releasing PLG vaccines resulted in statistically similarT cell and anti-tumor responses, they had differential effects on DCrecruitment and activation. In vitro tests indicated that GM-CSF was themost potent chemotactic factor for cDCs (FIGS. 23A-B). This finding wasconsistent with the ability of GM-CSF releasing matrices to recruitsignificantly more total DCs (˜10⁶, on par with ex vivo DC basedprotocols) and activated DCs, in comparison to FL scaffolds (FIGS. 24A-Cand 25A-C). In contrast, and in agreement with other reports, FLvaccines led to more matrix-resident pDCs when combined with CpG-ODNdanger signaling. pDCs are an important source of Th1 priming cytokinesthat amplify CTL responses, and increased pDCs numbers likelycontributed to the enhanced local production of IL-12 and IFNγ at thevaccine site. As described herein, GM and FL are combined in materialsystems to exploit GMCSF mediated activation of cDCs and FL mediatedgeneration of pDCs. This creates a superior DC network that is employedfor cancer vaccination and immunotherapy.

The results presented herein indicate that pDCs, and their cDCcounterparts are targeted to exploit their specialized abilities tomediate anti-tumor T cell responses. In contrast to nanoparticletargeting systems, the polymer systems described herein not only serveas an antigen delivery devices to recruit and activate DCs, but alsoserve as a physical structure where DCs temporarily reside while theyare activated.

The systems described herein demonstrated significant anti-tumoractivity. In addition to the polymers, e.g., PLG, described herein,matrices are optionally fabricated from other more inflammatory polymersto boost immune responses and DC mobilization. Another important aspectof subsequent T cell priming by these cells is LN homing. The exit ordispersement of DCs after antigen exposure is optimized by incorporatingdifferent adjuvants into the material to activate migratory function.Alternatively, other matrix properties, including degradation kineticsand porosity are altered to promote further control over DC trafficking.

Altogether, these findings provide evidence that FL, CCL20 and GM-CSFare utilized in biomaterial systems to mimic infection-inducedrecruitment of DCs in situ. As described herein, infection-mimickingporous devices are effective as therapeutic cancer vaccines.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-27. (canceled)
 28. A device comprising a polymeric structurecomposition, a tumor antigen, and a TLR3 agonist, wherein said TLR3agonist comprises condensed polyinosine-polycytidylic acid (poly I:C)cationic nanoparticles, said condensed poly I:C cationic nanoparticlesbeing formed by mixing said poly I:C with a polycation comprisingpositively charged amine groups at a charge ratio resulting inpositively charged condensates and an average particle size of 98-545nm.
 29. The device of claim 28, further comprising a TLR9 agonist. 30.The device of claim 28, wherein said TLR3 agonist is present at aconcentration effective to induce production of interleukin-12 (IL-12)by dendritic cells.
 31. The device of claim 28, wherein when the deviceis implanted in a subject, 200-400 ng/mL of IL-12 is produced at thesite of implantation.
 32. The device of claim 28, wherein the condensedpoly (I:C) cationic nanoparticle comprises PEI-poly(I:C).
 33. The deviceof claim 29, wherein the TLR9 agonist comprises a cytosine-guanosineoligonucleotide (CpG-ODN) or condensed CpG-ODN.
 34. The device of claim28, wherein the polymeric structure composition comprisespoly-lactide-co-glycolide (PLG).
 35. The device of claim 29, wherein thepolymeric structure composition comprises poly-lactide-co-glycolide(PLG).
 36. The device of claim 28, further comprising pathogenassociated molecular patterns (PAMPs).
 37. The device of claim 29,wherein the TLR9 agonist comprises a nucleic acid.
 38. The device ofclaim 37, wherein the nucleic acid comprises a cytosine-guanosineoligonucleotide (CpG-ODN) or a PEI-CpG-ODN.
 39. The device of claim 28,further comprising a recruitment composition.
 40. The device of claim39, wherein the recruitment composition comprises granulocyte macrophagecolony stimulating factor (GM-CSF), Flt3L, or CCL20.
 41. The device ofclaim 40, wherein the device comprises 0.5 μg to 500 μg of GM-CSF. 42.The device of claim 40, wherein the recruitment composition comprisesencapsulated GM-CSF.
 43. The device of claim 28, wherein the tumorantigen comprises a tumor lysate, purified protein tumor antigen, orsynthesized tumor antigen.
 44. The device of claim 28, comprising 100μg-10,000 μg of tumor antigen.
 45. The device of claim 28, wherein thetumor antigen is a lung cancer tumor antigen.
 46. The device of claim28, wherein the tumor antigen comprises an antigen from a cancerselected from the group consisting of a central nervous system (CNS)cancer, CNS Germ Cell tumor, Leukemia, Multiple Myeloma, Renal Cancer,Malignant Glioma, breast cancer, squamous cell carcinoma, ovariancarcinoma, prostate cancer, Kaposi's sarcoma, colon cancer,adenocarcinoma, testicular cancer, hepatocellular carcinoma, Synovialsarcoma, and Medulloblastoma.
 47. The device of claim 29, wherein thetumor antigen comprises an antigen from a cancer selected from the groupconsisting of a central nervous system (CNS) cancer, CNS Germ Celltumor, Leukemia, Multiple Myeloma, Renal Cancer, Malignant Glioma,breast cancer, squamous cell carcinoma, ovarian carcinoma, prostatecancer, Kaposi's sarcoma, colon cancer, adenocarcinoma, testicularcancer, hepatocellular carcinoma, Synovial sarcoma, and Medulloblastoma.48. The device of claim 29, further comprising a recruitmentcomposition.
 49. The device of claim 48, wherein the recruitmentcomposition comprises granulocyte macrophage colony stimulating factor(GM-CSF), Flt3L, or CCL20.
 50. The device of claim 29, wherein the tumorantigen comprises a tumor lysate, purified protein tumor antigen, orsynthesized tumor antigen.
 51. The device of claim 28, wherein saidpolymeric structure composition is anionic and said condensed poly I:Ccationic nanoparticles are electrostatically immobilized on said anionicpolymeric structure composition of said device.
 52. The device of claim29, wherein said polymeric structure composition is anionic and saidcondensed poly I:C cationic nanoparticles are electrostaticallyimmobilized on said anionic polymeric structure composition of saiddevice.
 53. The device of claim 28, wherein said charge ratio comprisesan amino-phosphate charge ratio of 3, 4, 7, or
 15. 54. The device ofclaim 29, wherein said charge ratio comprises an amino-phosphate chargeratio of 3, 4, 7, or
 15. 55. The device of claim 28, further comprisinga TLR7 agonist.
 56. The device of claim 55, wherein the TLR7 agonistcomprises a guanosine analog or an imidazoguinoline.
 57. The device ofclaim 56, wherein the TLR7 agonist comprises a guanosine analog, andwherein the guanosine analog is loxorbine.
 58. The device of claim 56,wherein the TLR7 agonist comprises an imidazoquinoline, and wherein theimidazoquinoline is imiquimod.